Method for treatment of antigenically modified polypeptides

ABSTRACT

Endogenous and exogenous proteins, and fragments thereof, are chemically modified outside the body of an animal so that when injected into the animal they produce more antibodies against the unmodified protein than would injection of the unmodified protein or fragment alone. The chemical modification may be accomplished by attaching the proteins or fragments to carriers such as, for example, bacterial toxoids. The chemical modification can also be accomplished by polymerization of protein fragments. Proteins which can be modified include Follicle Stimulating Hormone and Human Chorionic Gonadotropin. The modified polypeptide may be administered to animals for the purpose of contraception, abortion or treatment of hormone-related disease states and disease disorders, treatment of hormone-associated carcinomas, and to boost the animals&#39; resistance to exogenous proteins, for example viral proteins.

This application is a divisional application of pending application Ser.No. 07/935,331, filed Aug. 26, 1992, which is herein incorporated byreference, which is a continuation of Ser. No. 07/311,331, filed Feb.17, 1989, now abandoned, which is a divisional of application Ser. No.07/073,748, filed Jul. 15, 1987, now U.S. Pat. No. 5,006,334, which is acontinuation-in-part application of application Ser. No. 07/667,863,filed Nov. 2, 1984, now U.S. Pat. No. 4,691,006, which is acontinuation-in-part of both U.S. application Ser. No 06/472,190, filedMar. 4, 1983, now U.S. Pat. No. 4,526,716, and of international patentapplication PCT/U.S. No. 83/00777, filed May 18, 1983, theaforementioned international application, which names Vernon C. Stevensas the U.S. applicant is, insofar as regards the United States ofAmerica, a continuation-in-part of the aforementioned U.S. applicationSer. No. 06/472,190, now U.S. Pat. No. 4,526,716, which is itself acontinuation-in-part of application Ser. No. 06/323,690 filed Nov. 20,1981, now U.S. Pat. No. 4,384,995, which is a continuation-in-part ofapplication Ser. No. 06/112,628, filed Jan. 16, 1981, now U.S. Pat. No.4,302,386, which is a divisional of application Ser. No. 05/936,876,filed Aug. 25, 1978, now U.S. Pat. No. 4,201,770, which in turn is acontinuation-in-part of application Ser. No. 05/622,031, filed Oct. 14,1975, now abandoned, which in turn is a continuation-in-part ofapplication Ser. No. 05/462,955, filed Apr. 22, 1974, now abandoned,which in turn is a continuation-in-part of application Ser. No.05/406,821, filed Oct. 16, 1973, now abandoned, which in turn is acontinuation-in-part of application Ser. No. 05/357,892, filed May 7,1973, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to antigenic modification of polypeptides. Morespecifically, this invention relates to processes for modifyingpolypeptides which are not substantially immunogenic to the immunesystem of mammals so as to make the modified polypeptides moreimmunogenic. The invention also relates to the modified polypeptides soproduced, to vaccines containing such modified polypeptides, and forprocesses for affecting in various ways the metabolism of animals usingsuch modified peptides and vaccines.

It is well known that antibodies are generated in humans and in otheranimals in response to the presence of foreign antigens. It is alsoknown to confer immunity on an animal by administering an antibodyformed elsewhere. For instance, patents to Michaelson (U.S. Pat. No.3,553,317), Friedheim (U.S. Pat. No. 2,388,260), Reusser (U.S. Pat. No.3,317,400) and Peterson (U.S. Pat. No. 3,376,198) relate to productionof antibodies, which when injected into an animal of a different speciesor into a human being cause passive immunization. In patents to Fell(U.S. Pat. Nos. 2,301,532 and 2,372,066), the patentee refers to activeimmunization using modified histamine in such animals as horses, cows,etc. In a paper by R. G. Edwards in the British Medical Journal, Vol.26, pages 72 to 78, published in 1970, on "Immunology of Conception andPregnancy", he surveys the literature regarding the possibilities ofutilizing immunological methods to influence or control fertility,surveying first production of antibodies against testes or spermatozoa.Much of the literature surveyed is directed to the production of foreignantibodies which are injected into the subject (passive immunization).

Hormone antibodies have been studied for a long time and the effect ofspecific antisera have been recorded for many years. It is known thatadministration of certain antibodies during pregnancy can suppressimplantation or cause fetal resorption. Several different approacheshave been tried ranging from the induction of near permanent infertilityin the case of agglutination of spermatozoa in the male to thedisturbance of a single pregnancy by passive immunization withantibodies.

There are serious limitations to the use of passive immunizationprocedures for human therapy. Since the antibodies are practicallyproduced only in non-human animals, the repeated injection of animalproteins into humans is known to produce serious reaction in manyindividuals.

British Patent Specification No. 1,058,828 discloses that smallmolecules, referred to as "serological determinant peptides", can becoupled to large protein molecules, such as cattle albumin, and theresultant conjugate then may be injected into animals for antibodyproduction. The document lists proteins from which the serologicallydeterminant peptides may be isolated prior to being used in the processtaught, the collection including viruses and bacteria whose surfacecomponent has the characteristics of a protein, toxins and hormoneshaving protein structure and enzymes. No specific hormone is named inthe document and no utility of anti-hormone immunization is described.The patent specification references a publication entitled: "TheSpecificity of Serological Reactions", Dover Publications, Inc., NewYork, 1962, Chapter V, "Artificial Conjugated Antigens" by K.Landsteiner. This publication outlines various chemical methods andapplies them passively to bind various toxic substances in the bloodsuch as arsenic. Thyroxine data provided in the publication suggeststhat such methods may be applied to hormones without indicating thetherapeutic application, the publication teaching that specificantibodies may be formed to the small molecules and these antibodies arecapable of neutralizing the biological action of a large protein fromwhich the small peptide was a part.

Recently it has been discovered that doses of certain steroidsconsisting of synthetic non-protein hormones ("The Pill") whenadministered at stated intervals usually confer protection againstpregnancy for a short time (possibly a month). This medication hassometimes been found to create undesirable side effects in creatingundesirable metabolic changes and sometimes changes in the bloodclotting mechanisms. Moreover, the effect of each dose is of such shortduration that often it is of limited application, particularly in remoteareas to persons not readily instructed on proper and continuing use.

There is therefore a need for an effective safe method of creating atemporary but relatively one-time immunity against pregnancy which doesnot have serious side effects. There is also a need for an effectivesafe method of terminating a pregnancy soon after conception which doesnot have serious harmful side effects. Such need may be met by theneutralization of a reproductive protein which is necessary for thenormal events of conception and/or gestation.

There is also a need for a means for control of various disease statesor maladies caused or influenced by unusual excesses of certainpolypeptides such as gastrin, angiotension II, or somatomedian. It isbelieved that this invention meets this need safely and effectively.

There are also other medical needs which can be met by the presentinvention. It is well known to those skilled in the art of cancertreatment that certain cancers are highly resistant to the attack whichwould normally be made on the cancer by the immune system of a mammal inwhich the cancer is located. It is believed that this high resistance ofcertain cancers to attack by mammalian immune systems is due to theability of the cancer to coat its external surface with materials whichclosely resemble certain materials endogenous to the animal in which thecancer is located, so that the animal's immune system does not detectthe "foreign nature" of the cancer and hence is unable to attack it. Thepresent invention may provide a mechanism by which the pseudo-endogenousmaterial coating certain cancers can be stripped away, therebyfacilitating attack on such cancers by the immune system of the animalin which the cancer is located.

One further use for the processes, compositions and therapeutic methodsof the instant invention may be in dealing with diseases caused byagents which are not highly antigenic to mammalian immune systems.Although mammalian immune systems are extremely complex and extremelyeffective in dealing with most non-endogenous materials (such asbacteria and viruses) which find their way into the bodies of mammals,there are certain at least potentially dangerous non-endogenousmaterials which are not strongly antigenic to certain mammalian immunesystems, and which thus do not provoke a sufficiently strong responsefrom the immune system to avoid possible damage to the animal's body.The instant invention furnishes ways in which relatively non-antigenic,non-endogenous materials, for example vital proteins, can besynthetically modified to make them more strongly antigenic, therebyprovoking the formation, in the body of animals, of relatively largequantities of antibodies to the non-endogenous materials, withconsequent reduced risk of damage to the immune system if it thereafteris exposed to the non-endogenous materials.

SUMMARY OF THE INVENTION

As already indicated, this invention is concerned with processes for theproduction of modified polypeptides, with the modified polypeptides soproduced, with vaccines containing the modified polypeptides and withprocesses for the use of the modified polypeptides.

More specifically, this invention provides an antigen forimmunologically controlling biological activity in an animal byeliciting antibody formation, the antigen comprising carrier moietiesbiologically foreign to the animal and selected having a size sufficientto elicit antibody response non-harmful to normal body constituentsfollowing the administration thereof into the body of the animal, thesecarrier moieties being chemically conjugated with polypeptides having anamino acid sequence of the beta subunit of Chorionic Gonadotropin; andthe conjugate produced by the conjugation effecting a constitution oftwo or more immunological determinants effective to elicit antibodyresponse to the endogenous hormone, Chorionic Gonadotropin, upon theadministration thereof to the animal.

This invention also provides a process for preparing an antigen forprovoking the formation, in the body of an animal, of antibodies to aprotein which is not endogenous or immunogenic to said animal, theprocess comprising activating the protein, or a peptide having asequence corresponding to at least part of the sequence of the proteinand having a sulfhydryl group thereon by treatment with an activator ofthe structure A shown in FIG. 9 of the accompanying drawings, where Xrepresents a non-reacting group comprising substituted or unsubstitutedphenyl or C₁₋₁₀ alkylene moieties, or a combination thereof, or an aminoacid chain, so as to cause reaction of the activator with the sulfhydrylgroup, and treating the resultant activated protein or peptide with acarrier biologically foreign to the animal and selected having a sizesufficient to elicit antibody response following the administrationthereof into the body of the animal, the carrier having an amino groupthereon.

The invention also provides a process for preparing an antigen forprovoking the formation, in the body of an animal, of antibodies to aprotein which is not endogenous or immunogenic to the animal, theprocess comprising activating under neutral or acid conditions a carriernot having a sulfhydryl group but having an amino group with anactivator of the structure A shown in FIG. 9 of the accompanyingdrawings, in which X is defined above, so as to cause reaction of theactivator with the amino group, the carrier being biologically foreignto the animal and selected having a size sufficient to elicit antibodyresponse following the administration thereof into the body of theanimal, and treating the resultant activated carrier with the protein,or a peptide having a sequence corresponding to at least part of thesequence of the protein, the protein or peptide having a sulfhydrylgroup thereon.

The invention also provides a process for preparing an isoimmunogen forcontrolling biological action in an animal, this process comprisingactivating under neutral or acid conditions a carrier not having asulfhydryl group thereon but having an amino group with an activator ofthe structure B shown in FIG. 9 of the accompanying drawings, in which Xis as defined above, so as to cause reaction of the activator with theamino group, the carrier being biologically foreign to the animal andselected having a size sufficient to elicit antibody response followingthe administration thereof into the body of the animal, and treating theresultant activated carrier with a hormone endogenous to the animal,non-hormonal polypeptide endogenous to the animal, or a synthetic ornatural fragment of either, having a sulfhydryl group.

The invention also provides a process for preparing an antigen forprovoking the formation in the body of an animal of antibodies to aprotein which is not endogenous or immunogenic to the animal, thisprocess comprising activating the protein, or a peptide having asequence corresponding to at least part of the sequence of the proteinand having a sulfhydryl group thereon by treatment thereof with anactivator of the structure B shown in FIG. 9 of the accompanyingdrawings, in which X is as defined above, so as to cause the reaction ofthe maleiimide group of the activator with the sulfhydryl group on theprotein or peptide and so as to minimize reaction of the active estergroup on the activator with any amino group present on the protein orpeptide, and treating the resulting activated protein or peptide atslightly alkaline pH with a carrier moiety biologically foreign to theanimal and selected having a size sufficient to elicit antibody responsefollowing the administration thereof into the body of the animal.

The invention also provides a process for preparing an isoimmunogen forcontrolling biological activity in an animal, which process comprisesactivating under neutral or acid conditions a carrier not having asulfhydryl group but having an amino group with an activator of thestructure B shown in FIG. 9 of the accompanying drawings, in which X isas defined above, so as to cause reaction of the activator with theamino group, the carrier being biologically foreign to the animal andselected having a size sufficient to elicit antibody response followingthe administration thereof into the body of the animal thereof, andtreating the resultant activated carrier with a hormone endogenous tothe animal, or a synthetic or natural fragment of either having asulfhydryl group.

The invention also provides a process for preparing an isoimmunogen forcontrolling biological activity in an animal which comprises reacting acarrier biologically foreign to the animal, having a size sufficient toelicit antibody response following the administration thereof into thebody of the animal, and having an amino group, with an activator presentas an active ester of chloro-, dichloro-, bromo- or iodo-acetic acid soas to cause reaction of the activator with the amino group, therebyconverting the amino group to a group of the formula --NH.CO.T, where Tis a group of the formula CH₂ Cl, CHCl₂, CHBr₂, or CH₂ I, and treatingthe resulting activated carrier with a sulfhydryl group-containinghormone endogenous to the animal, subunit of the hormone, peptidefragment thereof or synthetically derived peptide having a sequenceanalogous to at least a portion of this subunit, thereby causingreaction between the group T and the sulfhydryl group such that thecarbon atom of the group T becomes bonded to the sulfur atom of thesulfhydryl group to form a thioether.

The invention also provides a process for preparing an isoimmunogen forcontrolling biological activity in an animal which comprises reacting ahormone endogenous to the animal, subunit of such a hormone, peptidefragment thereof or synthetically derived peptide having a sequenceanalogous to at least a portion of such subunit, not having a sulfhydrylgroup but having an amino group, with an activator present as an activeester of chloro-, dichloro-, bromo- or iodo-acetic acid so as to causereaction of the activator with the amino group, thereby converting theamino group to a group of the --NH.CO.T where T is defined above, andtreating the resultant moiety with a sulfhydryl group-containing carrierbiologically foreign to the animal and having a size sufficient toelicit antibody response following the administration thereof into thebody of the animal, thereby causing reaction between the group T and thesulfhydryl group such that the carbon atoms of the group T becomesbonded to the sulfur atom of the sulfhydryl group to form a thioether.

The invention also provides a process for preparing an antigen forprovoking the formation, in the body of an animal, of antibodies to aprotein which is not endogenous or immunogenic to the animal, thisprocess comprising reacting a carrier biologically foreign to the animaland having an amino group with an activator present as an active esterof chloro-, dichloro-, bromo- or iodo-acetic acid so as to causereaction of the activator with the amino group, thereby converting theamino group to a group of the formula --NH.CO.T, where T is as definedabove, and treating the resulting activated carrier with the protein, ora peptide having a sequence corresponding to at least part of thesequence of the protein, the protein or peptide having a sulfhydrylgroup, thereby causing the reaction between the group T and thesulfhydryl group such that the carbon atom of the group T becomes bondedto the sulfur atom of the sulfhydryl group to form a thioether.

The invention also provides a process for preparing an antigen forprovoking the formation, in the body of an animal, of antibodies to aprotein which is not endogenous or immunogenic to the animal, thismethod comprising reacting the protein or a peptide having a sequencecorresponding to at least part of the sequence of the protein, theprotein or peptide not having a sulfhydryl group but having an aminogroup, with an activator present as an active ester of chloro-,dichloro-, bromo- or iodo-acetic acid so as to cause reaction of theactivator with the amino group, thereby converting the amino group to agroup of the formula --NH.CO.T, where T is as defined above, andtreating the resulting moiety with a sulfhydryl group-containing carrierbiologically foreign to the animal, thereby causing reaction between thegroup T and the sulfhydryl group such that the carbon atom of the groupT becomes bonded to the sulfur atom of the sulfhydryl group to form athioether.

The invention also provides a method of controlling biological activityattributable to hormone and non-hormonal protein activity in an animal,which method comprises administering to the animal an immunologicallyeffective amount of a modified polypeptide, this modified polypeptide,consisting of a protein hormone, a non-hormonal protein, or a fragmentof either which has been chemically modified outside the body of theanimal, the protein hormone, non-hormonal protein or fragment having theproperties of (a) in unmodified form, being non-immunogenic to themammal and having a molecular structure similar to an endogenous proteinhormone or a non-hormonal protein, the biological function of which isdesigned to inhibit, or a fragment of either; and (b) in modified form,causing antibodies to be formed in the body of the mammal which inhibitthe biological function of the endogenous protein hormone ornon-hormonal protein following administration of the modified form intothe body of the mammal.

Examples of the control of biological activity attributable to hormoneand non-hormonal protein activity which can be achieved by this methodare as follows:

(1) control of Zollinger-Ellison Syndrome using modified polypeptidesderived from gastrin, or fragments thereof;

(2) control of hypertension using modified polypeptides derived fromangiotension I or II, or fragments thereof;

(3) control of elevated levels of growth hormone and/or somatomedianusing modified polypeptides derived from growth hormone, somatomedian orfragments of either of these hormones;

(4) control of kidney stones using modified polypeptides derived fromparathyroid hormone or fragments thereof;

(5) control of hyperinsulinoma using modified polypeptide derived frominsulin, glucagon or fragments of either of these hormones;

(6) control of hyperthyroidism using modified polypeptides derived fromthyroid stimulating hormone or fragments thereof; and

(7) control of irritable bowel syndrome using modified polypeptidesderived from secretin or a fragment thereof.

The invention also provides a vaccine for provoking the formation, inthe body of an animal, of antibodies to a protein which is notsubstantially immunogenic to the animal, this vaccine comprising amodified polypeptide of the invention derived from the protein or afragment thereof together with a vehicle, this vehicle comprising amixture of mannide monooleate with Squalane and/or Squalene.

The invention also provides a modified polypeptide for provoking theformation, in the body of an animal, of antibodies to a protein, themodified polypeptide comprising a linear polymer of polypeptidefragments, each of the fragments, in its monomeric form, beingsubstantially non-immunogenic to the animal and having a molecularstructure similar to a fragment of the protein to which antibodies areto be provoked, the linear polymer, after administration into the bodyof the animal, having a greater capacity to provoke the formation of theantibodies than the protein, the linear polymer being substantially freeof non-linear polymers of the fragments.

In another aspect, this invention provides a method for producing amodified polypeptide for provoking the formation, in the body of ananimal, of antibodies to a protein which is substantiallynon-immunogenic to the animal, the method being characterized by:

(a) procuring a first peptide having a molecular structure similar to afragment of the protein, the first peptide not having an unblocked thiolgroup and having an unblocked amino group only at its N-terminal but noother unblocked amino group;

(b) reacting the first peptide with an amino group activating agent,thereby producing an activated amino group at the N-terminal of thefirst peptide;

(c) reacting the activated first peptide with a second peptide having amolecular structure similar to a fragment of the protein, the secondpeptide having a C-terminal cysteine bearing an unblocked thiol groupbut not having any other unblocked thiol groups, thereby coupling theN-terminal of the first peptide to the C-terminal of the second peptide;

(d) reacting the resultant compound in a form having an unblocked aminogroup at its N-terminal but no other unblocked amino groups, and nounblocked thiol group, with an amino-group activating agent, therebyproducing an activated amino-group at the N-terminal of the resultantcompound;

(e) reacting the activated compound produced in step (d) with a furtherpeptide having a molecular structure similar to a fragment of theprotein, this further peptide having a C-terminal cysteine bearing anunblocked thiol group, but not having any other unblocked thiol groups,thereby coupling the activated N-terminal of the reactivated compoundproduced in step (d) to the C-terminal of the further peptide; and

(f) repeating steps (d) and (e) until the desired polymer length hasbeen achieved.

In another aspect, this invention provides an antigen for provoking theformation, in the body of an animal, of antibodies to a protein which isnot endogenous nor substantially immunogenic to the animal,characterized in that the antigen comprises the protein, or a peptidehaving a sequence corresponding to at least part of the sequence of theprotein, which protein or peptide has been chemically modified outsidethe body of the animal, the antigen having a greater capacity to provokethe formation of the antibodies than the protein in its unmodified form.

In another aspect, this invention provides a process for preparing anantigen of the invention, which process is characterized by:

procuring a protein which is not endogenous or immunogenic to theanimal, or the peptide having a sequence corresponding to at least partof the sequence of the protein; and

chemically modifying the protein or peptide outside the body of theanimal, thereby producing the antigen of the invention.

In another aspect, this invention provides a modified antigen for use infertility control in an animal characterized in that it comprises anantigen derived from the zona pellucida or from sperm, or a peptidehaving a sequence corresponding to at least part of the sequence of sucha zona pellucida or sperm antigen, which antigen or peptide has beenchemically modified outside the body of the animal, the modifiedantigen, after administration into the body of the animal, having agreater capacity to provoke the formation of antibodies than theunmodified antigen from which it is derived.

This invention also provides a peptide having an amino acid sequencecorresponding to the C-terminal sequence of the beta subunit of humanchorionic gonadotropin, the peptide comprising from 20 to 45 amino acidresidues.

Finally, this invention provides a method of controlling fertility in ananimal which comprises administering to the animal an immunologicallyeffective amount of a modified polypspride consisting of FSH, HCG, LH,HPL, prolactin, relaxin, an antigen derived from the zona psilucida orfrom sperm, or a fragment of any one of these hormones, which has beenchemically modified outside the body of the animal, the hormone orfragment having the properties of (a) in unmodified form, beingsubstantially non-immunogenic to the animal; and (b) in modified form,causing antibodies to be formed in the body of the animal, theseantibodies being capable of inhibiting the biological function of thehormone from which the modified polypeptide is derived.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart describing the results of mating four baboons threetimes following the administration thereto of a fertility controllingantigen according to the invention;

FIG. 2 shows two plots illustrating the antifertility antibody levelsmaintained within two baboons following the administration of antigensthereto formulated in accordance with the invention;

FIG. 3 shows three dose response lines illustrating the specificity ofantibody response to a CG antigen formulated in accordance with theinvention;

FIG. 4 shows the levels of antibody to beta-HCG produced in rabbits byvarious modified peptides in Example XXXII below;

FIG. 5 shows the levels of antibody to HCG produced in rabbits byvarious tetanus-toxoid-coupled modified peptides in Example XXXII below;

FIG. 6 shows the levels of antibody to HCG produced in rabbits byvarious tetanus-toxoid-coupled modified peptides having differingpeptide carrier ratios in Example XXXII below;

FIG. 7 shows the levels of antibody to HCG produced in rabbits byvaccines containing a tetanus-toxoid-coupled modified peptide andvarious adjuvants in Example XXXIII below;

FIG. 8 shows the levels of antibodies to HCG and peptide produced inmice of various species by a vaccine comprising a tetanus-toxoid-coupledmodified peptide coupled to various adjuvants in Example XXXIII below;

FIG. 9 shows the formulae of three coupling agents used to prepare themodified polypeptides of the invention; and

FIG. 10 shows typical reactions used to prepare conjugated modifiedpolypeptides of the invention, together with examples of the modifiedpolypeptides produced by such conjugation reactions.

DETAILED DESCRIPTION OF THE INVENTION

As will be apparent from the foregoing Summary of the Invention, theinvention is of extremely broad scope and is applicable to modificationof a large number of proteins, both endogenous and non-endogenous, andmodifications of fragments of such proteins. In view of the complexityof the invention, and the fact that many aspects of the invention, suchas the particular preferred modification techniques, do not vary greatlyfrom one protein or fragment to another, the following plan will beadopted in this Detailed Description both for brevity and for clarity.Following a general introduction, the various aspects of this inventionwill be discussed under three main headings. Firstly, this descriptionwill discuss the selection of the protein or fragment to be used toachieve a desired effect in a mammal. Secondly, the techniques used tomodify the peptides in order to increase the antigenicity thereof willbe discussed. Finally, the discussion will focus on the modes ofadministration of the modified polypeptides, including discussion of thevehicles used to carry such modified polypeptides and certain additiveswhich may be useful in conjunction with the modified polypeptides. Thissection of the discussion will also discuss appropriate modes ofadministration of the modified polypeptides.

One important aspect of this invention relates to the use of modifiedpolypeptides in actively immunizing an animal, particularly a mammal,against the biological action of endogenous unmodified hormone and/ornon-hormonal natural protein. The state of immunity (in the sense ofcausing the immune system of the animal to which the modifiedpolypeptide is administered to react against the larger proteinendogenous to the animal, whereas of course normally the immune systemwill not react to endogenous proteins) arises because of the creation ofantibodies which act against the antigenic modified polypeptide and itsendogenous, unmodified counterpart which is neutralized (renderedbiologically ineffectual) as a result of the existence of theantibodies. The immunity may take place because of the inability of theantibody to distinguish between the modified polypeptide and thenaturally existing protein, but it is uncertain that this is in fact thesituation. In effect, the invention provides, in one aspect, for theisoimmunization of a primate or other mammal.

For example, one important aspect of this invention, which is discussedin much more detail below, relates to the modification of proteinreproductive hormones by adding certain numbers of foreign moieties (orcarriers) to each hormone molecule, or hormone or fragment, orpolymerization of fragments of the relevant hormone. The modificationmust be sufficient to cause the body to create antibodies to themodified hormone which will neutralize or inhibit the biologicalactivity of the natural hormone produced by the body. Thus, the modifiedhormones become antigenic and cause the production of antibodies whichdisrupt the natural processes of conception and/or gestation. The term"protein reproductive hormones" includes those hormones essential to thenatural events of the reproductive process, including hormonesassociated with the production of sperm in the male as well as thoseassociated with the reproductive function of the female.

The immunochemical control (isoimmunization process), as already noted,neutralizes the naturally occurring hormone or the entity biologicallyanalogous thereto. As a consequence, the hormone or entity is no longeravailable as would normally be the case, for example, in the stimulationof some activity of a target tissue. Conversely, the neutralization ofthe biological activity of the hormone or analogous entity may serve totake away an inhibitory action which it otherwise might assert.

As indicated above, a theory leading to this invention was that thechemical modification of an essential reproductive hormone would alterit such that it would exhibit antigenic properties so that when injectedinto an animal (including humans) it would cause the formation ofantibodies which in turn would not only bind to the injected modifiedhormone but also to the natural unmodified endogenous hormone as well.With this theory in mind, reproductive hormones of various species weremodified and tested in baboons. The results illustrated that modifiedhormones of unrelated species do not produce the desired results,whereas modified hormones of the same or closely related species doproduce the desired results. It will accordingly be clear that thepolypeptide to be modified should be so related to the endogenoushormone or non-hormonal protein as to be either from the same animalspecies or be the immunological equivalent thereof as modified.

Additional experiments were conducted to test the validity of thisconcept in humans, i.e. modified human reproductive hormones wereinjected into humans. Collectively, the results prove the conclusiondrawn from the experiments with the baboons, namely, that isoantigenicimmunization using modified human reproductive hormones does producecontraception or interruption of gestation. Detailed examples whichfollow illustrate this result.

It is known that fragments of endogenous hormones exhibit essentially noantigenic properties. However, should a large enough fragment of anendogenous hormone be slightly modified as indicated above, thenantibodies will be formed which will react in the same way as if themodification is on a whole hormone, provided the large fragment issufficiently distinctive in chemical and physical make-up as to berecognized as a specific part of the whole.

Whether the hormone or specific fragment thereof is naturally occurringor is a synthetic product is clearly immaterial. A synthetic hormonemolecule will perform the same function as the naturally occurring one,being equivalent for the purpose of this invention. In this connection,it will be noted that certain natural substances with which thisinvention is concerned possess carbohydrate moieties attached at certainsites thereon whereas the corresponding synthetic polypeptides do not.Nevertheless, for the purpose of the instant specification and claims,the synthetic and natural polypeptides are treated as equivalents andboth are intended to be embraced by this invention. Reference in theabove regard is made to Table 3 herein as read in conjection withExample XXIX.

Thus, where the word "hormone" or "hormone molecule" is used herein, theword "synthetic" may be added before "hormone" without changing themeaning of the discussion. Similarly, the word "fragment" may beinserted after "hormone" or "molecule" without changing the meaning,whether or not "synthetic" has been inserted before "hormone".

The present invention is, however, not limited to modification ofprotein reproductive hormones, and numerous further examples ofmodification of the hormones and non-hormonal endogenous proteins willbe given below. Moreover, not merely is the invention applicable tomodification of non-reproductive endogenous proteins, the invention alsois applicable to modification of non-endogenous proteins. Although mostnon-endogenous proteins are to some extent immunogenic, theimmunogenicity of certain non-endogenous proteins, for example someviruses, is so low that the body of a mammal into which the virus entersmay fail to produce antibodies to the weakly immunogenic non-endogenousprotein in such quantities as to effectively remove the deleteriousnon-endogenous protein from the animal's system. Accordingly, themodification techniques of this invention may be employed to increasethe immunogenicity of non-endogenous proteins in order to ensure a moresatisfactory response from the immune system of the mammal, thereby ofcourse rendering the mammal much less prone to the deleterious effectsof the unmodified non-endogenous protein if the immune system is laterchallenged with such non-endogenous protein.

Whether the relevant hormone, non-hormonal protein, non-endogenousprotein or specific fragment thereof which is modified by the techniquesof the instant invention is naturally occurring or is a syntheticproduct is clearly immaterial. A synthetic protein molecule or peptidewill perform the same function as a naturally occurring one, inasmuch asthe body will react in an equivalent antigenic manner to both. It hasaccordingly been discovered by virtue of this invention that it ispossible to interfere with or treat various disease states or medicalproblems which are caused or influenced by certain polypeptides byactive immunization of a male or female animal by the production and useof antigens formed by administration of modified polypeptides. Themodification of the polypeptides forms antigens which are thenadministered into an animal in which immunization is desired.

The invention is useful for both the human and other animals. Similarly,although the main focus of the fertility control aspects of theinvention discussed in more detail below is on treating females, suchtechniques may be applicable to males e.g. modified polypeptides basedupon FSH, its beta subunit and fragments thereof, together with modifiedpolypeptides based upon sperm antigens or relaxin. Such immunizationrepresents an effective fertility control technique, provided nophysiological consequences are encountered which may be found to reactadversely to the performance of other body constituents.

It should be noted that the term "endogenous" is used herein to denote aprotein which is native to the species to be treated, regardless ofwhether the relevant protein, fragment or antigen is endogenous to theparticular individual animal being treated. Thus, for example, forpurposes of this application, a porcine sperm antigen is regarded asbeing endogenous to a sow even though obviously such a sperm antigenwill not normally be present in the body of a sow.

As already noted, the modified polypeptides of the invention which arederived from endogenous protein hormones, non-hormonal proteins orfragments thereof, provoke, when administered into the bodies ofappropriate mammals, antibodies to the endogenous proteins from whichthe modified polypeptides are derived. Consequently, not only can suchmodified polypeptides be used to influence the biological activity in amammal to which they are administered by generating antibodies to anendogenous protein in the mammal, but the modified polypeptides of theinvention (whether prepared by coupling the endogenous protein orfragment thereof to a carrier, or by coupling a plurality of suchfragments together) can also be used to generate antisera by introducingthe modified polypeptides into the body of a mammal, thereby provokingthe formation, in the mammal, of antibodies to the "endogenous protein";note that in such a method, since the modified polypeptide need not beintroduced into the same mammal, or even a mammal of the same species,as the animal from which it is derived or, in the case of a modifiedpolypeptide based upon a synthetic fragment, the mammal whose protein itmimics, the so-called "endogenous protein" used in this method need notbe endogenous to the mammal in which the antibodies are raised.

Following the raising of the antibodies in the mammal, some of theantibodies are recovered from the mammal, using conventional techniqueswhich will be familiar to those skilled in the art of immunology.Techniques generating monoclonal antibodies may also be used to generatethe desired antibodies. The antibodies thus generated can then be usedfor a variety of purposes. For example, such antibodies may be used forassaying the quantity of an endogenous protein in a mammal by bringingat least some of the recovered antibodies into contact with body tissueor body fluid from the mammal and observing the formation ornon-formation of the reaction process between the recovered antibody andthe endogenous protein indicative of the presence or absence of theendogenous protein in the body tissue or body fluid assayed. If, in thismethod, the endogenous protein assayed is one associated with pregnancy,this assay method can function as a pregnancy test. If, on the otherhand, the endogenous protein assayed is one the presence or absence ofwhich is associated with reduced fertility or infertility in the mammalfrom which the body tissue or body fluid is derived, the assay canfunction as a test for reduced fertility or infertility in such amammal.

SELECTION OF POLYPEPTIDE FOR MODIFICATION

As already indicated, the present invention is applicable to almost anyhormonal or other protein related activity in a mammal, and toactivities, such as infections, in mammals caused by non-endogenous butrelatively weakly immunogenic protein agents, such as vital proteins.Examples of natural hormones and natural non-hormonal proteins which maybe modified according to this invention include Follicle StimulatingHormone (FSH), Luteinizing Hormone (LH), Luteinizing Hormone ReleasingHormone (LH-RH) , relaxin, Chorionic Gonadotropin (CG) , e.g. HumanChorionic Gonadotropin (HCG), Placental Lactogen, e.g. Human PlacentalLactogen (HPL), Prolactin, e.g. Human Prolactin (all of which areproteinaceous reproductive hormones), Gastrin, angiotension I and II,growth hormone, somatomedian, parathyroid hormone, insulin, glucagon,thyroid stimulating hormone (TSH), secretin, and other polypeptideswhich could adversely affect body function.

Despite the very wide range of proteins to which the techniques of thepresent invention can be applied, there are certain considerations whichshould always be borne in mind when considering the selection of anappropriate polypeptide for modification by the techniques of theinstant invention. Firstly, it is of course necessary to determine whichhormone or combination of hormones or other protein is responsible forthe condition or problem which it is desired to treat. However, in manycases this will still leave one with a large number of possible proteinswhich could be modified by the techniques of the instant invention. Forexample, if one wishes to use the instant invention to render a femalemammal infertile, one can approach the problem by modifying FSH, LH,LH-RH, CG, PL, relaxin or a variety of other protein hormones which areknown to be involved in the female mammalian reproductive system. Oneimportant consideration which should always be borne in mind in choosinga polypeptide for modification by the instant invention is the problemof cross-reactivity. As well known to those skilled in the field ofimmunology, it is not uncommon to find that antibodies intended to reactwith one protein (the "target" protein) also react to a significantextent with other, non-target proteins. This is a serious problem, sinceit may cause the administration of a modified polypeptide intended toprovoke the formation of antibodies to one specific natural hormone tocause the generation of antibodies to one or more other hormones, whichit is not desired to effect. In some cases, the reactions with thenon-target proteins may cause damage to essential body functions.Accordingly, so far as possible the peptide selected for modification bythe instant invention should be chosen so that the modified polypeptidewill provoke, in the body of the mammal to be treated, the formation ofantibodies which are highly specific to the target protein.

Although in some cases, especially where the target protein isrelatively small (for example LH-RH or angiotension I or II), it may bein practice essential to modify the whole target protein, since afragment comprising less than the whole target protein, will, even whenmodified by the instant techniques, fail to provoke sufficient antigensto the target protein, in general, especially when dealing withrelatively complex target proteins such as insulin or HCG, the use of afragment of the target protein rather than the intact target protein isrecommended for use in modification according to the instant invention.As already mentioned, it is well recognized by those skilled inimmunology (see e.g. W. R. Jones, "Immunological Fertility Regulation",Blackwell Scientific Publications, Victoria, Australia (1982) (theentire disclosure of this work is herein incorporated by reference),pages 11 et. seq. that one of the greatest potential hazards of avaccine, especially a contraceptive vaccine, is cross-reactivity withnon-target antigens, producing what is essentially anartificially-induced autoimmune disease capable of causingimmunopathological lesions in, and/or loss of function of, the tissuescarrying the cross-reactive antigens. Two possible mechanisms for suchcross-reactivity are:

(a) presence of shared antigenic determinants; a complex protein maycontain components (amino-acid sequences) identical to those present innon-target antigens; and

(b) steric overlap between non-identical but structurally related partsof the target and non-target antigens. Obviously, the threats posed byboth these modes of cross-reactivity may be lessened by using, in themodified polypeptides of the invention, a fragment of a complex proteinrather than the whole protein. Since the fragment has a simplerstructure than the protein from which it is derived, there is lesschance of shared antigenic determinants or steric overlap withnon-target antigens. In particular, cross-reactions cannot be avoided byusing fragments derived from a portion of the target protein which isnot similar in sequence to the non-target but cross-reactive antigen. Totake one specific example, one of the major problems in provokingantibodies to HCG is cross-reactivity of HCG antibodies with LH, thiscross-reactivity being at least largely due to virtual identity of aminoacid sequence between LH and the 1-110 amino acid sequence of the betasubunit of HCG. Accordingly, when it is desired to form an HCG-derivedmodified polypeptide of the invention, the fragment used is preferablyone having a molecular structure similar to part or all of the 111-145sequence of the beta subunit of HCG, since it is only this 111-145sequence of beta-HCG which differs significantly from the correspondingsequence of LH.

Thus, in most cases the polypeptide modified by the techniques of theinstant invention is preferably a fragment of the target protein ratherthan the intact target protein. More accurately, one should use as thepolypeptide to be modified by the techniques of the invention a fragmenthaving a molecular structure similar to a fragment of the targetprotein. In saying that the fragment has a molecular structure similarto a fragment of the target protein, it is not necessarily implied thatthe entire amino acid sequence of the fragment must correspond exactlyto part of the sequence of the target protein; for example, in certaincases some substitution of amino acids may be possible without effectingthe immunogenic character of the fragment. For example, theaforementioned U.S. Pat. No. 4,302,386 describes a polypeptide,designated Structure (IX) (which is also discussed in detail below),which is notionally derived from the beta subunit of HCG but in whichthe cysteine residue at the 110-position is replaced byalpha-aminobutyric acid. Furthermore, it is shown in the examples belowthat although the natural form of the beta subunit of HCG contains anumber of carbohydrate residues attached to the amino-acid chain,synthetic peptides corresponding in sequence to the relevant parts ofthe HCG sequence, but lacking such carbohydrate residues, can bemodified by the techniques of the instant invention and give goodresults.

Although species specificity is of course a consideration in anyimmunological process, I do not exclude the possibility of the fragmentsmodified by the instant processes may actually be derived from a proteinof a different species of mammal than the mammal to which they are to beadministered, since many proteins are either identical between speciesor differ from one another so little in amino acid sequence thatconsiderable cross-reactivity exists between antibodies to thecorresponding proteins of the two species. For example, as mentionedbelow, zona pellucida enzymes from a pig will, when injected intohumans, produce antibodies which display considerable activity againsthuman zona antigens. Accordingly, for example, if one wishes to form amodified polypeptide for provoking the formation of antibodies, inhumans, to zona pellucida antigens, appropriate polypeptide fragmentsmay be prepared from the zona pellucida antigens of pigs. Also, thefragments modified by the instant processes may incorporate sequences ofamino acids having no counterpart in the sequence of the protein fromwhich the fragment is notionally derived. Again, for example, it isshown below that one may use in the instant processes certainpolypeptide fragments, designated Structures (IV), (VIII), (IX), (X) and(XIV) which are notionally derived from the beta subunit of HCG butwhich incorporate spacer sequences comprising multiple proline residues.

Of course, one should be cautious when using sequences not exactlycorresponding to portions of the target protein. For example, theprotein relaxin is known to be highly species specific and accordinglyit is not recommended that fragments of non-human relaxin proteins bemodified by the instant methods and injected into humans to provoke theformation of anti-relaxin-antibodies in humans.

In choosing an appropriate polypeptide for modification according to theinstant invention, amino-acid sequence is, however, not the only factorwhich has to be considered; it is also necessary to pay close attentionto the conformation, that is to say the physical shape, of the proteinor fragment selected for modification relative to the naturalconformation of the target protein. It is well known to those skilled inthe art of immunology that the conformation or shape of an antigen is animportant factor in allowing recognition of the antigen by an antibody.Accordingly, if a polypeptide modified according to the instantinvention does not retain the conformation of the relevant part of thetarget protein, it is likely that the antibodies provoked by injectionof the modified polypeptide into a mammal will not display optimumactivity against the natural target protein. For example, a peptidehaving the same sequence as part of the target protein will probably notwork very well if, because of the absence of other parts of the sequenceof the target protein which affect the positioning of the crucialantigenic determinant in the natural target protein, the fragment usedto prepare the modified polypeptide of the invention adopts aconformation very different from the conformation of the same amino acidsequence in the target protein. Similarly, because of the way in whichthe chain of a complex target protein will normally be folded, theantigen-antibody binding reaction may rely upon recognition of two ormore amino acid sequences which are widely separated along the chain ofthe target protein but lie, in the natural conformation of the targetprotein, closely adjacent one another in space. All these considerationsmay enter into the question of what is the most appropriate polypeptideto use in the instant invention.

As those skilled in the art are aware, one major factor effecting theconformation, and hence the antigenic properties and antigenicdeterminants, of complex proteins is the presence of cysteine residuesand disulfide bridges in such proteins. It is well known to thoseskilled in the art that, in many natural proteins containing cysteineresidues, these residues are not present in their thiol form containinga free --SH group; instead, pairs of cysteine residues are linked bymeans of disulfide bridges to form cystine. Such disulfide bridges arevery important in determining the conformation of the protein. In mostcases, the disulfide bridges present in the natural form of the proteinare easily reduced to thiol groups by means of mild reducing agentsunder conditions which leave the remaining parts of the protein moleculeunchanged. Such breaking of disulfide bridges causes major changes inthe conformation of the protein even though no disturbance of the aminoacid sequence occurs. In particular, the twelve cysteine residuespresent in the beta subunit of HCG are, in the natural form of thesubunit, coupled together to form six disulfide bridges, so that thenatural form of the protein has no free thiol groups. (It should benoted that the exact manner in which the twelve cysteine residues areinterconnected to form the six disulfide bridges is not at presentknown, although the location of three of the six bridges has been madewith reasonable certainty.)

The generation of free thiol groups by reduction of disulfide bridges innaturally occurring forms of proteins may affect the cross-reactivity ofthe antibodies produced when a modified polypeptide derived from theprotein or a fragment thereof is injected into an animal. As alreadymentioned, an antibody frequently recognizes its corresponding antigennot only by the amino acid sequence in the antigen but also by theconformation of the antigen. Accordingly, an antibody which binds verystrongly to a protein or a peptide in its natural conformation may bindmuch less strongly, if at all, to the same protein or polypeptide afterits conformation has been drastically altered by breaking disulfidebridges therein.

Accordingly, the breaking of disulfide bridges in proteins or otherpolypeptides may provide a basis for reducing the cross-reactivitybetween antibodies to antigens having the same amino acid sequence alongparts of the molecule. For example, it has been pointed out above thatcross-reaction is frequently encountered between antibodies to beta-HCGand HLH because the first 110 residues in the beta-HCG and HLH sequenceare virtually identical in the natural forms of the two molecules, thusthe conformations are also presumably very similar. It has beensuggested above that one means of producing in an animal antibodies tobeta-HCG which do not substantially cross-react with HLH is to supply tothe animal an antigen of the invention derived from a polypeptide whichcontains all or part of the residues 111-145 of beta-HCG but which lacksall or substantially all of the residues 1-110 of beta-HCG. In effect,this approach avoids antibody cross-reaction with HLH by chemicallyremoving from the modified polypeptide of the invention the sequence ofresidues which is common to beta-HCG and HLH. As an alternativeapproach, by cleaving the appropriate number of disulfide bridges in thenatural form of beta-HCG, it may be possible to so alter theconformation of residues 1-110 thereof that the antibodies formed when amodified polypeptide of the invention based upon thisaltered-conformation beta-HCG is administered to an animal will nolonger cross-react significantly with HLH. In other words, instead ofchemically severing the common sequence of residues from beta-HCG inorder to prevent cross-reaction, it may be possible to leave this commonsequence of residues in the beta-HCG but to so alter the conformation ofthis common sequence that, to an antibody, the altered-conformationcommon sequence does not "look" like the natural form of the commonsequence, so that an antibody which recognizes the altered-conformationcommon sequence will not recognize the natural-conformation commonsequence in HLH. Moreover, once the natural conformation of the sequenceof residues 1-110 has been destroyed by breaking the disulfide bridges,this common sequence will probably assume the helical conformationcommon in polypeptides lacking disulfide bridges, so that this part ofthe beta-HCG will not be strongly immunogenic and most of the antibodiesformed by an antigen of the invention based upon thealtered-conformation beta-ECG will be antibodies to the sequence 111-145which is not common with HLH. Obviously, cross-reactivity betweenantibodies to other pairs of hormones may similarly be destroyed byaltering the conformation of portions of the two proteins which aresimilar and hence will otherwise promote antigen cross-reactivity.

Appropriate polypeptides derived from certain important proteins andsuitable for modification by the instant processes will now be discussedin more detail. However, it is stressed that the following specificapplications of the instant processes are not limitative, since asalready explained the present invention is applicable to modification ofpolypeptides derived from a very wide variety of both endogenous andnon-endogenous proteins.

REPRODUCTIVE HORMONES

As is well known to those skilled in the art, the hormone systemaffecting reproduction in both male and female mammals is extremelycomplex, and the instant invention may be used to control fertility inboth males and females by interference with a very wide variety ofhormones. At present, the preferred polypeptides for modification by theinstant processes are polypeptides derived from CG, polypeptides derivedfrom zona pellucida or sperm antigens or placental antigens, andpolypeptides derived from relaxin. Each of these three groups ofpolypeptides will now be discussed individually.

CHORIONIC GONADOTROPIN

The hormone, Chorionic Gonadotropin (CG) has been the subject ofextensive investigation, it being demonstrated in 1927 that the bloodand urine of pregnant women contained a gonad-stimulating substancewhich, when injected into laboratory animals, produced marked gonadalgrowth. Later, investigators demonstrated with certainty that thePlacental Chorionic villi, as opposed to the pituitary, were the sourceof this hormone. Thus, the name Chorionic Gonadotropin or, in the caseof humans, Human Chorionic Gonadotropin (HCG) was given to this hormoneof pregnancy. During the more recent past, a broadened variety ofstudies have been conducted to describe levels of HCG in normal andabnormal physiological states, indicating its role in maintainingpregnancy. The studies have shown the hormone's ability to induceovulation and to stimulate corpus luteum function, and evidence has beenevoked for showing its ability to suppress lymphocyte action. Theimmunological properties of the HCG molecule also have been studiedwidely. Cross-reaction of antibodies to HCG with human pituitaryLuteinizing Hormone (LH), and vice-versa, has been extensivelydocumented, see for example:

Paul, W. E. & Ross, F. T., "Immunologic Cross Reaction Between HCG andHuman Pituitary Gonadotropin", Endocrinology, 75, 352-358 (1964);

Flux, D. X. & Li, C. H., "Immunological Cross Reaction AmongGonadotropins",Acta Endocrinologic, 48, 61-72 (1965);

Bagshawe, K. D.; Orr, A. H. & Godden, J., "Cross-Reaction inRadio-Immunoassay between HCG and Plasma from Various Species", Journalof Endocrinology, 42, 513-518 (1968);

Franchimont, P., "Study on the Cross-Reaction between HCG and PituitaryLH", European Journal of Clinical Investigation, 1, 65-68 (1970);

Dorner, M.; Brossmer, R.; Hilgenfeldt, U. and Trude, E, "Immunologicalreactions of Antibodies to HCG with HCG and its chemical derivatives inStructure-Activity Relationships of Proteins and Polypeptide Hormones",(ed. M. Margoulies & F. C. Greenwood), pp 539, 541 Amsterdam: ExcerptaMedica Foundation (1972);

Further, these cross-reactions have been used to perform immunoassaysfor both CG and LH hormones. See:

Midgley, A. R. Jr., "Radioimmunoassay: a method for HCG and LH",Endocrinology, 79, 10-16 (1966);

Crosignani, P. G.; Polvani, F. & Saracci, R., "Characteristics of aradioimmunoassay for HCG-LH; in Protein and Polypeptide Hormones" (ed.M. Margoulies) pp. 409, 411 Amsterdam: Excerpta Medica Foundation(1969);

Isojima, S.; Nake, O.; Kojama, K. & Adachi, H., "Rapid radioimmunoassayof human L. H. using polymerized antihuman HCG as immunoadsorbent",Journal of Clinical Endocrinology and Metabolism, 31, 693-699 (1970).

Although the entire CG hormone or a subunit thereof, for example thebeta submit, may be modified by the instant processes, in general it ispreferred to use a polypeptide corresponding to only a fragment of thebeta subunit. More specifically, as already noted there is a largeportion of the beta subunit of CG which is almost identical to thecorresponding beta subunit of LH, so that it is desirable to use afragment corresponding to a portion of the 111-145 sequence of the betasubunit of CG which is not common to LH, thereby avoiding thecross-reactivity of CG and LH antibodies already discussed above. Thus,an immunological reaction against the hormone CG can be achieved withoutcausing undesirable immune reactions to the naturally occurring bodyconstituent LH. Synthetic polypeptides corresponding to the desiredfragments of CG offer enhanced practicality both from the standpoint ofproduction costs and the high degree of purity needed for commercial usein a contraceptive maxim.

Subunits and fragments of the proteinaceous reproductive hormonesinclude the beta subunit of natural Follicle Stimulating Hormone, thebeta subunit of natural Human Chorionic Gonadotropin, fragmentsincluding, inter alia, a 20-30 or 30-39 amino acid peptide consisting ofthe C-terminal residues of natural Human Chorionic Gonadotropin betasubunit, as well as specific unique fragments of natural Human Prolactinand natural Human Placental Lactogen, which may bear little resemblanceto analogous portions of other protein hormones. Further with respect tothe type of novel chemical entities with which this invention isconcerned, one may note for instance the chemical configuration of thebeta subunit of HCG. That structure is as follows: ##STR1##

For specificity of antibody action it is necessary that distinctivepeptides be isolated or prepared that contain molecular structurescompletely or substantially completely different from the otherhormones. The beta subunit of HCG possesses a specific chain or chainsof amino acid moieties which differ either completely or essentiallyfrom the polypeptide chain of Human Luteinizing Hormone. These chains orfragments, when conjugated with a carrier, represent an additionalaspect of this invention. Accordingly, the polypeptide Structures (II)and (III) C-terminal portions of structure I! ##STR2## whether obtainedby purely synthetic methods or by enzymatic degradation from the naturalor parent polypeptide, Carlson et al., J. Biological Chemistry, 284(19), 6810, (1973)! when modified according to this invention, similarlyprovide materials with antigenic properties sufficient to provide thedesired immunological response.

The beta subunit set forth at structure (I) is seen to represent achemical sequence of 145 amino acid components. This structure has ahigh degree of structural homology with the corresponding subunit ofLuteinizing Hormone (LH) to the extent of the initial 110 amino acidcomponents. As indicated above, it may be found desirable, therefore toevoke a high specificity to the Chorionic Gonadotropin hormone or ananalogous entity through the use of fragments analogous to theC-terminal, 111-145 amino acid sequence of the subunit. Structure (II)above may be observed to represent just that sequence. Structure (III)is slightly shorter, representing the 116-145 amino acid positionswithin the subunit sequence.

Further polypeptide chains useful for modification by the instantprocesses to promote antibody build-up against natural CG include thefollowing structures labeled Structures (IV)-(XIV). When modified by theinstant processes, these polypeptide provide immunogenic activityagainst HCG. All of these polypeptides are considered fragments of HCGby virtue of their substantial resemblance to the chemical configurationof the natural hormone and the immunological response provided thereprovided by them when modified by the instant processes. ##STR3##

Structure (IV) will be recognized as incorporating a Cys component atthe amino or N-terminal which is associated with a proline spacersequence. These spacers serve to position the sequence which followsphysically distant from the carrier-modifier. The latter sequence may beobserved to present the 138th to 145th amino acid components sequence ofthe subunit Structure (I). Structure (V) on the other hand, representsan initial sequence corresponding with the 111th to 118th components ofthe subunit Structure (I) followed by a sequence of six proline spacercomponents and a carboxyl terminal, present as cysteine. The rationalein providing such a structure is to eliminate the provision of siteswhich may remain antigenically neutral in performance. Structures (IV)and (V) represent relatively shorter amino acid sequences to the extentthat each serves to develop one determinant site. Consequently, asalluded to in more detail hereinafter, they are utilized in conjunctionwith a mixed immunization technique wherein a necessary two distinctdeterminants are provided by the simultaneous administration of two suchfragments, each conjugated to a corresponding, separate carriermacromolecule. Structure (VI) represents the 115th through 145thcomponent sequence of structure (I). Structure (VII) represents aportion of Structure (I), however, essentially, a sequence of the 111thto 130th components thereof is formed.

Structure (VIII) incorporates two sequences, one which may be recognizedin Structure (V) and the other in Structure (IV). These two sequencesare separated by two spacer sequences of proline components and one isjointed with an intermediately disposed cysteine component which servesa conjugation function as described later herein. With this arrangement,two distinct determinant sites are developed in physically spacedrelationship to avoid the development of an unwanted artificialdeterminant possibly otherwise evolved in the vicinity of their mutualcoupling. Structure (VIIIa) represents Structure (VIII) with additionalproline spacer residues to provide a widened spacing of determinantsites.

Structure (IX) mimics sequences from Structure (I) with the addition ofa proline spacer sequence, a cysteine component at the C-terminal, andan Aba substituted for cysteine at the 110 position. The Aba designationis intended herein to mean alpha aminobutyric acid of Cysteine.Structure (X) will be recognized as a combination of Structure (II) witha six residue proline spacer sequence and a cysteine component at theC-terminal. Similarly, Structure (XI) combines Structure (II) with acysteine component at the C-terminal without a proline spacer sequence.##STR4##

Structure (XII) will be recognized as having the sequence of Structure(II) with the addition of Thr-Cys residues at its N-terminal. Structure(XIII) is similar to Structure (IX) but does not contain the spacersequence. Structure (XIV) will be recognized as being similar toStructure (II) with the addition of spacer components at the N-terminaland a cysteine residue, which may be useful for modification reactions,as described in more detail below.

As already mentioned, it is only the 111-145 amino acid sequence ofbeta-HCG which differs from the corresponding sequence of LH. However,research indicates that the polypeptides used in the instant processesmay contain sequences corresponding to the 101-110 sequence which iscommon to beta-HCG and beta-LH without inducing the formation ofantibodies reactive to LH. Thus, one can use in the instant antigens andmethods peptides containing part or all of the common 101-110 sequencewithout causing substantial cross-reactivity with LH. For example,Structure (II) above represents the 111-145 amino acid sequence ofbeta-HCG. If desired, therefore, a peptide having the 101-145 amino acidof beta-HCG could be substituted for the peptide of Structure (II) inthe instant modified polypeptides without substantially affecting theactivity of the modified polypeptide and without causingcross-reactivity with beta-LH.

Two further preferred polypeptides derived from beta-HCG are primarilyintended for use in the linear polymers of polypeptides discussed inmore detail below. These two preferred fragments are: ##STR5##

As mentioned earlier, the main utility presently envisaged for modifiedpolypeptide of the invention derived from mammalian reproductivehormones or fragments thereof is useful as contraceptives and/orabortifactants by administration of the modified polypeptide to thefemale mammal. However, modified polypeptides of the invention derivedfrom mammalian reproductive hormones or fragments thereof have a varietyof other uses. Since the modified polypeptides do provoke the productionof antibodies to the endogenous reproductive hormone when injected intoanimals, they can be used, in ways which will be familiar to thoseskilled in the art, for the production of antibodies specific to theendogenous reproductive hormone from which the modified polypeptide isderived. The antibodies may be produced, for example, by injecting themodified polypeptide into a suitable mammal, extracting blood or otherbody fluid or tissue from the mammal and harvesting the antibodies fromthe extracted blood, body fluid or tissue.

The antibodies thus produced may be used in a wide variety of tests andtreatments. For example, since the antibodies thus produced are specificto an endogenous hormone, they may be used, in ways which will befamiliar to those skilled in the art, to perform qualitative orquantitative assays for the endogenous hormone in the tissues or bodyfluids of the mammal which produced the endogenous hormone to which theantibody is specific, the antibodies produced by the process of thepresent invention may be useful in diagnostic tests to determine whetherhormone levels in a mammal are abnormal. For example, abnormal levels,usually lowered levels, of certain reproductive hormones are oftenassociated with reduced fertility or infertility in man and othermammals, and consequently antibodies produced by the processes of theinvention may be used in tests for such conditions of reduced or absentfertility. Such tests for reduced or absent fertility are not onlyuseful in humans, but may also be desired by veterinarians charged withthe care of valuable breeding animals such as stallions at stud orvaluable pedigree bulls. For example, a peptide having a requisitesequence determinant of equine chorionic gonadotropin can be used toprepare a modified polypeptide of the invention, which can then bepassed through a suitable mammal to generate antibodies to equinechorionic gonadotropin. Such antisera would be useful for infertilitydiagnosis in valuable thoroughbred horses.

The antibodies produced by the process of the present invention may alsobe useful in pregnancy tests in man and other mammals. As previouslynoted, human chorionic gonadotropin was first discovered because it ispresent at relatively high levels in the urine of pregnant women, anddetection of the elevated levels of human chorionic gonadotropin in theurine of pregnant women is the basis for most pregnancy tests. By virtueof their specificity to human chorionic gonadotropin (or thecorresponding gonadotropin in other mammalian species) antibodiesproduced by the process of the present invention may be useful in suchpregnancy tests. Such pregnancy tests are not only useful in humans; forexample, a pregnancy test may be highly desirable in a brood mare inorder to ensure that she is in foal. In the absence of such a pregnancytest, an owner might incur an additional heavy stud fee unnecessarily.

The uses of the antibodies produced by the processes of the presentinvention are not, however, confined to assay of the level of endogenoushormones (or, if desired, non-hormonal proteins) in mammals. Inaddition, the instant antibodies may also be useful in producingphysiological changes in the tissues of a mammal.

POLYPEPTIDES DERIVED FROM THE ZONA PELLUCIDA, FROM SPERM OR FROMPLACENTAL TISSUE

Another group of polypeptides which can be altered by the instantprocesses, and used in the field of fertility control in both humans andother mammals, are specific non-hormonal protein antigens isolated fromplacental tissue. There is direct evidence that inhibition of substancesthat are specific to the placental tissue and do not have similarantigenic properties with other antigens from organs in other parts ofthe body, can result in the disruption of pregnancies by passiveimmunization. Such specific placental substances when modified to formmodified polypeptides by the procedures described herein can be injectedinto the body of an animal of the same species as an effective fertilitycontrol means with the mechanism being active immunization similar tothat described for the antigenic modification of hormones. Theparticular advantages of these substances is that placental antigens areforeign to the non-pregnant female human subject and therefore areunlikely to cause any cross-reaction or disruption of normal bodyfunction in the non-pregnant female.

A further group of polypeptides which may be modified by the instantprocesses to yield modified polypeptides useful for fertility controlare polypeptides derived from zona pellucida or from sperm, and peptideshaving a sequence corresponding to at least part of the sequence of sucha zona pellucida or sperm antigen.

It is known that antigens from the zona pellucida (the outer covering ofthe ovum) when injected into female primates produce antibodies havinganti-fertilization effects, including prevention of sperm attachment to,and penetration of, the zona pellucida of the unfertilized ovum, andprevention of dispersal of the zona pellucida of the fertilized ovumprior to implantation (such dispersal of the zona apparently being anessential prerequisite for implantation). See e.g. W. R. Jones,"Immunological Fertility Regulation", Blackwell Scientific Publications,Victoria, Australia (1982), pages 160 et seq. Such anti-fertilityeffects are believed to be due to formation of an antibody-antigenprecipitate on the zona, this precipitate rendering the zona unable toundergo its normal sperm-binding reaction and also rendering the zonainsensitive to the action of the proteases normally responsible fordispersal of the zona.

Another possible approach to the production of anti-fertility vaccineuses sperm antigen. Several antigens, especially sperm enzymes, known toexist in sperm, may be used in the modified antigens of this invention;see W. R. Jones, op. cit., pages 133 et seq. The most promising suchantigen is the lactate hydrogenase known as LDH-C4 or LDH-X. Although ofcourse lactate dehydrogenases are present in other tissues, LDH-C4 isdistinct from other lactate dehydrogenase isoenzymes and appears to besperm-specific. Moreover, the enzyme is not strongly species specific,and methods for its isolation and purification are known. Again, thebest results should be obtained by modifying LDH-C4 or a fragmentthereof to produce a modified polypeptide of this invention. Severalnatural peptide fragments of LDH-C4 have been prepared, sequenced andshown to bind to antibodies against the parent molecule. (See E.Goldburg, "LDH-X as a sperm-specific antigen", in T. Wegmann and T. J.Gill (eds.), Reproductive Immunology, Oxford University Press, 1981).(The disclosure of this work is herein incorporated by reference.)

Although theoretically an anti-fertility vaccine based on sperm antigensmight be useful in males, the likelihood of testicular damage renders itmore likely that such a vaccine will find its utility in females. It isknown that circulating antibodies in the female bloodstream do penetratethe genital fluids; for example experiments in baboons with vaccinesbased upon the peptide of Structure (XII) above conjugated with tetanustoxoid have shown the presence of HCG antibodies in the genital fluids.However, one possible problem with any vaccine based on sperm antigensis maintaining a sufficiently high antibody level in female genitalfluids to complex with the large amounts of sperm involved.

RELAXIN

Another group of peptides which can be modified by the methods of theinstant invention for use in fertility control are relaxin andpolypeptides derived therefrom. It has been known for a long time thatrelaxin is a peptide hormone synthesized in the corpus luteum of ovariesduring pregnancy and the hormone is released into the bloodstream priorto parturition. The major biological effect of relaxin is to remodel themammalian reproductive tract to facilitate the birth process, primarilyby relaxing the cervix, thereby assisting in the dilation of the cervixprior to parturition. The amino acid sequence, which bears someresemblance to that of insulin, has been determined; see: Hudson et al.,"Structure of a Genomic Clone Encoding Biologically Active HumanRelaxin". This paper also gives methods for the synthesis of certainrelaxin-derived peptides.

The use of relaxin or peptides derived therefrom in fertility controlaccording to the instant invention depends not upon the natural functionof relaxin during parturition, but upon the fact that anti-relaxinantibodies are known to render sperm immotile. Thus, there appears to bea relaxin-like antigen present on the surface of sperm, especially sincethe immotility of the sperm can be reversed by adding relaxin to theantibody/sperm complex. As mentioned above, in theory one could usemodified sperm antigens prepared by the instant processes to generate inthe male antibodies to various antigens present in sperm, but there isthe serious problem that, owing to the blood/testes barrier, suchanti-sperm antigens do not penetrate the testes. The potentially veryrapid induction of immotility of anti-relaxin antibody rendersgeneration of such an antibody in males a highly attractive potentialform of male contraception. Although the anti-relaxin antibodies willnot penetrate the testes because of the blood/testes barrier, they canpenetrate the epididymis and they will also be secreted into the fluidwhich becomes mixed with the sperm shortly before or during ejaculation.Thus, by producing anti-relaxin antibodies in the male, ejaculationwould take place normally but the sperm produced would be immotile.Furthermore, the risk of complications and unintended tissue damage bysuch an instant process is slight, since the antibodies will not enterthe testes, thereby avoiding potentially damaging reactions due toantibody-antigen binding within the testes.

It should be noted that injection of modified relaxin-derived peptidesmodified by the instant processes into females is not recommended; sucha process would carry too great a risk of ovarian damage in the female.

It should also be noted that relaxin is a highly species-specificprotein. Accordingly, when choosing an appropriate peptide derived fromrelaxin for modification by the instant processes, care should be takento ensure that the peptide corresponds to part of the sequence of humanrelaxin (or, of course, relaxin of any other species which it isdesigned to treat).

CANCER TREATMENT

Another health problem that can be treated by the instant methods isthat of certain endocrine or hormone-dependent breast tumors or cancers.Certain of these cancers have been shown to be dependent upon theabundant secretion of the hormone prolactin for their continuedsurvival. The inhibition of the secretion of prolactin has been shown todiminish the growth rate and the actual survival of certain of thesetumors. The immunization of mammals suffering from such tumors withmodified polypeptides related to prolactin and produced by the instantmethods would result in the systematic reduction of the level ofprolactin circulating in the system and consequently may result in theregression or remission of tumor growth. The consequence of thistreatment would be far more favorable in terms of effective treatment ofthis disease, since surgical removal of the breast is the principalmethod of treatment currently available. It will of course be understoodthat this aspect of the instant invention will be effective only withregard to those tumors which are dependent upon the secretion ofprolactin (or some other hormone modifiable by the processes of theinstant invention) for survival.

Investigators have also determined, for example, that certainpolypeptide entities are supportive factors to, and secretions of,neoplastic diseases in both man and other mammals. These supportiveentities have biochemically, biologically and immunologically closeresemblances to hormones, particularly to CG as well as to LH. Byapplying the iso-immunization techniques of the instant invention (i.e.by injecting into the mammal a modified polypeptide which producesantibodies to a natural hormone or other protein of the mammal) thefunction of such polypeptides or endogenous counterparts can beneutralized to carry out regulation of the malignancy. For example,tumors in both male and female primates may be treated byisoimmunization procedures developing antibodies to CG or LH or thesupported entity analogous thereto. Furthermore, neoplasms in primatefemales may be regulated by isoimmunization procedures developingantibodies to endogenous LH. This hormone, when associated with a tumorstate, tends to aggravate the tumorous condition.

It appears (although the invention is in no way limited by this belief)that certain carcinomas exude CG or an immunologically-similar materialon their surfaces, thereby presenting to the immune system of the hostanimal a surface which, superficially, appears to be formed of materialendogenous to the host animal and which is thus relativelynon-immunogenic. Because of this known association between certaincarcinomas and CG or CG-like materials, the instant modified polypeptidederived from CG described above are useful not only for fertilitycontrol but also for treatment of carcinomas associated with CG orCG-like materials.

Example XXXIV below shows that a beta-HCG/tetanus toxoid modifiedpolypeptide of the invention confers upon rats substantially completeprotection against an injection of tumor cells of the virulent ratmammary adenocarcinoma R 3230 AC, which is associated with CG-likematerial. The aforementioned polypeptide of the invention, when givenprior to injection of a dose of tumor cells which causes 100% mortalityin unprotected, reduces the mortality to 0. Further work showing the useof the instant modified polypeptides in carcinomas is given in ExamplesXXXVI-XXXVIII.

HYPERTENSION

Another serious medical problem which can be treated by the instantinvention is that of hypertension. In general terms, the state ofhypertension is the abnormal level or fluctuation of one's bloodpressure. The blood pressure of an individual is controlled by manyphysiological processes in the body. However, two major substancesaffecting the regulation of such pressure are the hormonal polypeptidesknownas angiotension I and II. In certain states of high blood pressure(hypertension) it is difficult to control medically the secretion into,and therefore the level of angiotension I and II in, the circulatorysystem. By appropriate modification of one or both of these hormones andsubsequent immunization of the hypertensive patient with the modifiedhormone, it is possible to reduce the secretion of angiotension I and/orII in patients with chronically elevated hormone levels. The predictableand controlled reduction of these substances is beneficial to certainpatients with chronic problems of hypertension. Modified angiotension Iand II can be produced by any of the modification techniques describedbelow. The resultant modified angiotension I or II is simply injectedinto the patient in an amount sufficient to induce added antibodyresponse sufficient to control or regulate unmodified angiotension Iand/or II to the desired degree.

The structures of both angiotension I and II are known, that ofangiotension I being as follows:

    Asp-Arg-Val-Try-Ile-His-Pro-Phe-His-Leu,

while the structure of angiotension II is:

    Asp-Arg-Val-Try-Ile-His-Pro-Phe.

In view of the relatively small sizes of these peptides (the molecularweight of the I form is 1296.7 and that of the II form 1046.3), it isrecommended that modification be carried out using the intact hormone asthe polypeptide to be modified.

DIABETES AND ASSOCIATED VASCULAR DISEASES

The present invention is applicable to the treatment of diabetes andassociated micro- and macro-vascular diseases. Currently, the treatmentof diabetes is limited to dietary and/or drug treatment to regulateblood glucose levels. Recent scientific data support the concept thatgrowth hormone and somatomedian (both polypeptides) are intimatelyinvolved in the disease syndrome. These substances can be modified bythe technique described herein and used in an effective amount tocontrol the progress of this disease. In practice, modified growthhormone or modified somatomedian is injected into the body to developantibodies for control of the normally secreted hormones.

Naturally, the use of the present invention to treat elevated levels ofgrowth hormone and/or somatomedian is not confined to diabetic patients.Thus, the present invention may be used to treat non-diabetic patients,such as persons suffering from acromegaly, who have excessive levels ofgrowth hormone and/or somatomedian.

MISCELLANEOUS HORMONE-RELATED CONDITIONS

The present invention, as already noted, is applicable to the treatmentof an extremely wide range of hormone-related condition. Indeed, asexplained above, in principle the present invention is applicable to thetreatment of any condition which is caused by excessive levels of ahormone or non-hormonal protein in a mammal. One such disease statewhich can be treated by the present invention is the digestive disorderknown to those skilled in the medical field as Zollinger-EllisonSyndrome. This syndrome or disease state is generally described as acondition in which a hypersecretion of the polypeptide gastrin, which isproduced in the pancreas, brings about a state of hyperacidity in thestomach which results in a chronic digestive disorder. Heretofore, theonly effective treatment for this disease state was the surgical removalof a part or total removal of the subject's stomach. Although survivalof such patients is usually not threatened, the medical state and lifestyle of such individuals is severely affected by such treatment.

Treatment of such subjects with gastrin-derived modified polypeptides ofthe invention can be used to enhance the production of antibodiesagainst the hypersecretion of gastrin and thereby alleviate or reducethe systems of this disease without surgical intervention. Sufficientreduction by immunological means of this substance in the system of thebody would be sufficient to avoid the complicated and seriousconsequences of the surgical treatment currently in use. In practice, aneffective amount of modified gastrin is simply injected into the patientas required to accomplish the control of the flow or presence ofgastrin.

Other disease states and the associated hormones which may be modifiedby the instant processes or immunological treatment of the diseases areas follows:

(1) modified parathyroid hormone for the treatment of kidney stones,

(2) modified insulin and/or glucagon for the treatment ofhyperinsulinoma,

(3) modified thyroid stimulating hormone (TSH) for the treatment ofhyperthyroidism, and

(4) modified secretion for the treatment of irritable syndrome.

NON-ENDOGENOUS PROTEINS

In the specific aspects of the invention described above, the peptidewhich is modified to produce the instant modified polypeptide is apeptide having a sequence corresponding to at least part of the sequenceof a protein endogenous to the animal in which the modified polypeptideis to be employed. However, the techniques of the present invention canusefully be extended to proteins which are not endogenous norsubstantially immunogenic to the mammal to be treated. By the instantmethods, these substantially non-immunogenic, non-endogenous proteins orfragments thereof can be modified so as to stimulate the animal's immunesystem to produce antibodies to the non-endogenous proteins. As thoseskilled in the art are aware, there are numerous pathogens and similarmaterials known which are not endogenous to animals, which are capableof producing harmful effects in the animal's body but which are notimmunogenic to the animal, in the sense that introduction of thepathogen or other material into the body of the animal fails to elicitfrom the animal's immune system production of the quantity ofappropriate antibodies necessary for the animal's immune system todestroy the pathogen or similar material.

For example, the Herpes simplex Type II virus is capable of producing anumber of harmful effects in man, including the production of painfullesions in the genital areas. Although this virus has, like mostviruses, protein included in its structure, the viral protein is notstrongly immunogenic in most human beings, so that only about 50% ofinfected human beings produce antibodies to the virus. This lack ofimmune response to the virus by many human beings means that the viruscan remain in the infected human beings for at least several years, andthis persistence of the virus in the infected individuals not onlycauses these individuals to suffer recurrent attacks of the painfulsymptoms caused by the virus, but also renders them long-term carriersof the virus. This persistence of the virus in infected individuals isone of the factors largely responsible for the epidemic proportionswhich Herpes simplex infections have reached in several countries. Bypreparing an antigen of this invention derived from a protein having asequence similar to that of at least part of the sequence of a Herpessimplex vital protein, it is possible to stimulate the human immunesystem so as to render it capable of producing large quantities ofantibodies to the Herpes simplex virus. Not only should this stimulationof the immune system reduce the occurrence of symptoms associated withHerpes simplex infection, but it should help to control the spread ofthe virus. Similarly, the immune response of humans and other animals toviruses such as colds, influenza and other viruses can be increased bypreparing modified antigens of the invention based upon peptides havingsequences corresponding to vital proteins of the appropriate virus. If,as appears likely, a virus is responsible for acquired immune deficiencysyndrome (AIDS) a modified antigen of this invention could also be usedto produce immunity to this disease.

TECHNIQUES FOR MODIFICATION OF POLYPEPTIDES

A wide range of techniques may be used in the present invention tomodify the polypeptides. In general, any type of chemical modification,which renders the modified polypeptide more immunogenic to the mammal towhich it is to be administered than the unmodified polypeptide fromwhich the modified polypeptide is derived, may be used in the presentinvention. However, the two major chemical techniques of chemicalmodification employed in the present invention are conjugation of thepeptide to a similar carrier molecule, and polymerization (a term whichis used in its broad sense to include, for example, dimerization) of thepolypeptide. These two major techniques will now be discussed, andthereafter a group of miscellaneous chemical modification techniqueswhich may be useful in some instances will also be discussed. Many ofthe techniques described below are not in themselves novel and some ofthe techniques may be found in the following list of literaturereferences, while various others may be found elsewhere in literature bypersons skilled in the art:

1. Klotz et al., Arch. of Biochem. and Biophys, 96, 60, 605-612, (1966).

2. Khorana, Chem. Rev., S3, 145 (1953).

3. Sela et al., Biochem. J., 85, 223 (1962).

4. Eisen et al., J. Am. Chem. Soc., 75, 4583 (1953).

5. Centeno et al., Fed. Proc. (ABSTR), 25, 729 (1966).

6. Sokolowsky et al., J. Am. Chem. Soc., 86, 1212 (1964).

7. Tabachnick et al., J. Biol. Chem., 234, 1726, (1959).

8. Crampton et al., Proc. Soc. Exper. Biol. & Med., 80, 448 (1952).

9. Goodfriend et al., Science, 144, 1344 (1964).

10. Sela et al., J. Am. Chem. Soc., 78, 746 (1955).

11. Cinader et al., Brit. J. Exp. Pathol., 36, 515 (1955).

12. Phillips et al, J. of Biol. Chem., 240 (2), 699-704 (1965).

13. Bahl, J. of Biol. Chem., 244, 575 (1969).

It will be appreciated by those skilled in the art that, in the instantinvention, the chemical modification of the polypeptide is effectedoutside the body of the animal prior to introduction of the modifiedpolypeptide into the body of the animal.

In general, the methods used for preparing the instant modifiedpolypeptide based upon non-endogenous proteins, such as viral proteinsor peptides corresponding to parts thereof, are the same as those usedfor modifying endogenous proteins or fragments thereof, although it willbe appreciated that the preferred methods used for modifying anon-endogenous materials may differ in certain respects from those usedin modifying endogenous polypeptides. Since, in general, thenon-endogenous peptide will provoke at least a limited immune responsefrom the animal in which the antigen is to be administered, it may wellbe that the requirements for modification of the non-endogenous peptideto produce the instant modified polypeptide are less stringent thanthose modifications of an endogenous and completely non-immunogenicpolypeptide. However, since the non-endogenous polypeptide is beingmodified to increase its immunogenic effect in the animal into which itis to be administered, in general it will still be desirable that thecarrier used to modify the non-endogenous polypeptide to produce theinstant modified polypeptide be a material which itself provokes astrong response from the animal's immune system.

For example, where the modified polypeptide is prepared by conjugationof the polypeptide to a carrier, the carrier may be a bacterial toxoidsuch as diphtheria toxoid or tetanus toxoid.

CONJUGATION OF THE POLYPEPTIDE TO A CARRIER

One preferred way of effecting the necessary chemical modification ofthe polypeptide (whether that polypeptide be an intact protein or afragment thereof) in the processes of the instant invention is so-calledconjugation of the polypeptide to a carrier. Such conjugation isaccomplished by attaching to the polypeptide one or more foreignreactive (modifying) groups and/or by attaching two or more polypeptidesto a foreign reactive group (i.e. a carrier) or both of the above, sothat the body of the animal, recognizing the modified polypeptide as aforeign object, produces antibodies which neutralize not only themodified polypeptide but also the unmodified protein related to thepolypeptide and responsible for the disease or medical problem beingregulated.

For example, the HCG-derived peptide of Structure (II) may be modifiedby conjugation with a polytyrosine chain or a protein macromoleculecarrier, thereby causing the peptide of Structure (II) to becomeantigenic so that the resulting administration of the modified peptideof Structure (II) will provide the desired immunological action againstnatural HCG. Another example of chemical modification by conjugationwould be conjugation of any of the HCG-derived peptides designatedStructures (II)-(XIV) above by coupling to a carrier such as Ficoll 70(a synthetic copolymer of sucrose and epichlorohydrin having an averagemolecular weight of 70,000±1,000, good solubility in water, Stokesradius about 5.1, and stable in alkaline and neutral media, availablefrom Pharmacia Fine Chemicals, Pharmacia Laboratories, Inc. 800Centennial Avenue, Piscataway, N.J. 08854) or other carriers such as theprotein macromolecules described below.

Particularly where the larger whole hormone or subunit type molecularstructures are concerned, the number of foreign reactive groups whichare to be attached to the polypeptide and the number of polypeptides tobe attached to a foreign reactive group depends on the specific problembeing treated. Basically, what is required is that the concernedpolypeptide be modified to a degree sufficient to cause it to beantigenic when injected in the body of the animal. If too littlemodification is effected, the body may not recognize the modifiedpolypeptide as a foreign body and would not create antibodies againstit. If the number of foreign molecules added to the polypeptide is toogreat, the body will create antibodies against the intruder antigen, butthe antibodies will be specific to the injected antigen and will notneutralize the action of the concerned natural endogenous hormone ornon-hormonal protein, i.e. they will be specific to the modifier.

In general, again considering the larger molecule subunit or wholehormone, it has been found that about 1-40 modifying groups per moleculeof polypeptide will be useful in modifying the polypeptide adequately soas to obtain the desired immunological effect of this invention. As willbe appreciated by one skilled in the art, this ratio of modifying groupsper polypeptide will vary depending upon whether an entire hormone isutilized for modification or whether for instance a relatively smallsynthetic fragment of the hormone is to be modified. Generally for thelarger molecules, it is preferred that 2-40 modifying groups permolecule of polypeptide be used according to this invention. In theinstance where the polypeptide is the beta-subunit of HCG, it isparticularly preferred that about 5-30 and more preferably 10-26modifying groups per molecule of polypeptide be used. The importantconsideration with respect to each modified polypeptide is that thedegree of modification be adequate to induce generation by the animal ofantibodies adequate to neutralize some of the natural hormone ornon-hormonal protein the neutralization of which is desired, and thiswill vary with each polypeptide involved, and the degree of correctionor change desired for the body function involved.

Modification of the polypeptide is accomplished by attaching variouskinds of modifying groups to proteinaceous hormones, non-hormonalproteins, subunits or specific fragments thereof according to methodsknown in the art.

As will be apparent from the formulae given above, the HCG-derivedpolypeptides of Structures (II)-(XIV) are relatively smaller fragmentsof HCG, which can be produced synthetically. To render them capable ofeliciting antibody production, it becomes necessary to conjugate themwith larger carrier-modifier molecules. Generally about 5-30 peptidefragments will be coupled with one carrier molecule. The body will, ineffect, recognize these foreign carriers as well as the sequencesrepresented by the fragments and form antibodies both to the carrier andto the sequences of the coupled fragments. Note that thecarrier-modifiers are foreign to the body and thus antibodies to themwill not be harmful to any normal body constituents. In the latterregard, it may be found preferable to utilize a carrier which, throughthe development of antibodies specific to it, will be found beneficialto the recipient.

As indicated earlier herein, it also is preferred that the modificationconstitute two or more immunological determinants represented on thenative protein as polypeptide structures to which it is desired to evokean antibody response. The effect is one of heterogeneity of antibodydevelopment. Thus, several fragment structures have been described abovehaving at least two distinct amino acid sequences represented in the HCGbeta subunit. These sequences may be so spaced as to derive thedeterminants in mutual isolation, while the spaced sequence fragment isconjugated with a larger, macromolecular carrier. Alternately, the notedmixed immunization arrangement may be utilized wherein a first fragmentdeveloping one determinant is conjugated with a first carrier moleculeand is administered in combination with a second, distinct fragmentwhich is conjugated with a second carrier molecule, the latter of whichmay be the same as or different in structure from the first carrier.Thus, each macromolecular carrier must be conjugated with hormonefragments such that each fragment represents two or more immunologicaldeterminants. These two necessary determinants can be evolved by mixing,for example, separate conjugate structure, for example based uponStructures (IV) and (V) each of which, through forming antibodiesseparately to the distinct determinants, will provide a population ofantibodies reacting with two separate determinants on the naturalendogenous hormone.

Inasmuch as the noted fragments are relatively small as compared, forinstance, to a whole hormone or subunit thereof, a criterion of size isimposed upon the selection of a carrier. The carrier size must beadequate for the body immune system to recognize its foreign nature andraise antibodies to it. Additionally, carrier selection preferably ispredicated upon the noted antibody heterogeneity requirement, i.e. thecarrier must itself evoke a heterogeneous immune response in addition tothe fragments. For example, improved response may be recognized wherethe carrier is varied in structure, e.g. incorporating branching chainsto enhance the recognition of both the carrier and the attachedpolypeptide as being of a foreign nature.

As one example of whole hormone modification, modified diazo groupsderived from sulfanilic acid may be attached to the subjectpolypeptides, see the Cinader et al. and Phillips et al. referencescited above for instruction on how this "attachment" is accomplished,and to the extent necessary for an understanding of this invention, suchis incorporated herein by reference.

Additional modifying groups for modifying whole hormones or theirsubunits are those groups obtained by reaction of the polypeptides withdinitrophenol, trinitrophenol, and S-acetomercaptosuccinic anhydride,while suited for utilization as carrier-modifiers in conjunction withfragments, are polytyrosine in either straight or branched chains,polyalanines in straight or branched chains, biodegradable polydextrans,e.g. polymerized sugars such as sucrose copolymerized withepichlorohydrin, e.g. Ficoll 70 and Ficoll 400 (a synthetic copolymer ofsucrose and epichlorohydrin having an average molecular weight of400,000±100,000 intrinsic viscosity of 0.17 dl/g. specific rotationalpha!²⁰ _(D) of +56.5°, available from Pharmacia Fine Chemicals,Pharmacia Laboratories, Inc. 800 Centennial Ave., Piscataway, N.J.08854) or a polyglucose such as Dextran T 70 (a glucan containingalpha-1,6-glucosidic bonds and having an average molecular weight ofapproximately 70,000, synthesized microbiologically by the action ofLeuconostoc mesenteroides strain NRRLB-512 on sucose), serum proteinssuch as homologous serum albumin, hemocyanin from Keyhole limpet (amarine gastropod mollusk) viruses such as influenza virus (type A, B, orC) or poliomyelitis virus, live or killed, Types 1, 2 and 3 of tetanustoxoid, diphtheria toxoid, cholera toxoid or somewhat less preferably,natural proteins such as thyroglobulin, and the like. Generally,synthetic modifiers are preferred over the natural modifiers. However,carrier-modifiers found particularly suitable for conjugation with theabove-discussed fragment structures are flagellin, tetanus toxoid and aninfluenza subunit, for example, the preparation of which is described byBachmeyer, Schmidt and Liehi, "Preparation and Properties of a NovelInfluenza Subunit Vaccine", Post-Graduate Medical Journal, (June, 1976),52, 360-367. This influenza subunit was developed as a vaccine whichincorporates essentially only the two viral proteins, hemagglutinin andneuraminidase. Containing substantially only these two essentialimmunogens, the subunit represents a preparation which does not containother protein and lipid antigens which may be found to causeunderdesired side reactions. A secondary benefit may be realized throughthe utilization for example, of the influenza subunit, poliomyelitisvirus, tetanus toxoid, diphtheria toxoid, cholera toxoid or the like asa modifier-carrier, inasmuch as beneficial antibodies will be raised tothat modifier-carrier as well as the hormonal fragment conjugatedthereto.

Flagellin is a protein described as forming the wall of the main spiralfilament of the flagellum. Bacterial flagella, in turn, have been knownas the active organelles of cell locomotion, individual flagella(flagellum) occurring in suspension as individual spirals which, upondrying, collapse into filaments which describe a sine wave with a wavelength of 2-3 microns and an amplitude of 0.25-0.60 microns. Generally,the flagellum consists of three morphologically distinct parts: a basalstructure that is closely associated with the cytoplasmic membrane andcell wall, a hook and the noted main spiral filament.

Purified flagellin is readily obtained by solubilization of flagellarfilaments below a pH value of about 4, and subsequent removal of theinsoluble material by centrifugation or filtration. As a group ofrelated proteins, flagellins from different bacterial species have beenpredicted to have similar amino-acid compositions. However, the aminoacid composition of each flagellin species is unique. Essentially allflagellins are described as containing no or only a few residues ofcysteine, tryptophan, tyrosine, proline and histidine. Thus, whenconjugated with fragments in accordance with the invention, athiolactonization procedure or the like is carried out as describedlater herein.

The molecular weights of various flagellins have been calculated, in allcases the values thereof of the monomeric subunits falling in the rangeof 30,000 to 50,000. From an immunological standpoint, a flagellinmolecule is highly immunogenic. For further and more detailed discoursedescribing bacterial flagella and flagellin, reference is made to"Advance in Microbial Physiology", 6, 219(1971), "Bacterial Flagella" byR. W. Smith and Henry Coffler, which publication is incorporated hereinby reference.

Tetanus toxoids have been the subject of study and production for manyyears. The toxoid generally is evolved from a formalinization of tetanustoxin, the latter being a protein synthesized by Clostidium tetani.Immunization currently is carried out utilizing soluble and absorbedtetanus toxoid and suggestions have been made concerning the utilizationof fluid tetanus toxoid in complex with antitoxin. Publicationsdescribing the toxin and toxoid are numerous, reference being made tothe following:

1. "Immunochemistry of Tetanus Toxin", Bizzini, et al., Journal ofBiochemistry, 39, 171-181 (1973).

2. "Early and Enhanced Antitoxin Responses Elicited with Complexes ofTetanus Toxoid and Specific Mouse and Human Antibodies", Stoner et al.,Journal of Infectious Diseases, 131, (3), 230-238 (1975).

3. "Differences in Primary and Secondary Immunizability of Inbred MiceStrains", Ipsen, Journal of Immunology, 83, 448-457 (1959).

4. "Antigenic Thresholds of Antitoxid Responses Elecited in IrradiatedMice with Complexes of Tetanus Toxin and Specific Antibody", Hess etal., Radiation Research, 25, 655-667 (1965).

5. "Early and Enhanced Germinal Center Formation and Antibody Responsesin Mice After Primary Stimulation with Antigen-isologous AntibodyComplexes as Compared with Antigen Alone", Laissue et al., Journal ofImmunology, 107, 822-825 (1971).

6. "Distinctive Medullary and Germinal Center Proliferative Patterns andMouse Lymph Nodes after Regional Primary and Secondary Stimulation, withTetanus Toxoid", Buerki et al., Journal of Immunology, 112, (6),1961-1970 (1974).

Although the conjugation techniques have been mostly described above,and will in general be mostly described below, with reference toconjugation of polypeptides derived from natural protein hormones, itwill be appreciated that exactly similar techniques will be employed formodification of non-hormonal proteins or fragments thereof, for exampleviral proteins.

Methods for preparing the modified polypeptides of this invention alsoinclude the following.

In one preferred modification process, the polypeptide fragment would bemodified, for example that designated Structure (XII) above, isactivated first, after which it is conjugated with a carrier, forexample the influenza subunit described above, tetanus toxoid orflagellin. An activating reagent may be utilized which exhibitsdiffering functionality at its ends and, by choice of reactionconditions, these end functions can be made to react selectively. Forexample, the activators of Formulae A and B shown in FIG. 9 of theaccompanying drawings, which each have a maleiimido group and asubstituted acid group, may be used. In these activators, X is anon-reacting group which can be a substituted or unsubstituted phenyl orC₁ -C₁₀ alkylene moiety, or a combination thereof. The substituent onthe phenyl ring (if any) should of course be non-interfering with thereactions of the activator, as is the remainder of the grouping X.

The grouping X may be, inter alia, a pentamethylene, 1,4-phenylene ormonomethyl-1,4-phenylene grouping.

The maleiimido grouping of the above activators will react withsulfhydryl (SH) groups in the polypeptides to be modified underconditions whereby the opposite end (active ester end) of the reagentdoes not react with the amino groups present in the polypeptides. Thus,for example, polypeptides, such as that designated Structure (XII)above, contain a cysteine amino acid, and hence an SH group, react asshown in Equation 1 in FIG. 10 of the accompanying drawings. Followingthe above reaction, upon adjusting the pH to slightly alkalinecondition, for example, pH 8, and adding a carrier protein, conjugationis accomplished to produce the product of Formula 2 shown in FIG. 10 ofthe accompanying drawings.

Preferably a carrier protein, such as the above-noted flagellin, whichdoes not contain SH groups, but does contain NH₂ groups, may first betreated with an activator of the formula A or B shown in FIG. 9 of theaccompanying drawings, wherein X is as defined above, at pH 7 or lowerto cause reaction of the active ester end of the activator with theflagellin, giving a compound of Formula III shown in FIG. 10 of theaccompanying drawing. Following the above, the activated carrier isreacted with a polypeptide fragment containing an SH group to derive aproduct similar to that discussed immediately above.

Should the polypeptide fragment not contain an SH group, e.g. Structures(II), (III), (VI) and (VII), such structures can be modified first tointroduce such a grouping by standard methods such as"thiolactonization", following which they are conjugated utilizing theabove-discussed selective bi-functional reagents. For a more detaileddescription of these reagents, reference is made to the followingpublications:

O. Keller and J. Ridinger, Helv. Chim. Acta, 58, 531-541 (1975).

W. Trommer, H. Kolkenbrock and G. Pfleiderer, Hoppe-Seyler's Z. Physiol.Chem., 356, 1455-1458 (1975).

Further description of preferred embodiments of the above-describedutilization of bi-functional reagents is provided hereinbelow atExamples XXVII and XXVIII.

As already mentioned, in many natural proteins containing cysteineresidues, these residues are not present in the thiol form containing afree SH group; instead, pairs of cysteine residues are linked by meansof disulfide bridges to form cysteine. Accordingly, when it is desiredto produce free SH groups in proteins to carry out the couplingreactions discussed above, one convenient way of providing such free SHgroups may be to cleave disulfide bridges naturally present in theprotein or other polypeptides which it is desired to conjugate. Forexample, as noted above the natural form of beta-HCG contains sixdisulfide bridges. To produce free thiol groups for coupling reactions,any number of these bridges from 1 to 6 may be broken using knowntechniques as set out for example in:

Bahl et al, Biochem. Biophys. Res. Comm., 70, 525-532 (1976). Thisparticular article describes cleavage of 3-5 of the six disulfidebridges in beta-HCG, but the same techniques may be used to break allsix bridges if this is so desired. It should, however, be noted that thetechniques disclosed in this paper are not selective and although it ispossible to control the degree of disulfide bridge breaking, it is notpossible to break specific bridges and leave others; the breaking ofbridges is at random and the thiol groups produced are randomlydistributed over the possible positions in beta-HCG.

As an alternative approach to the utilization of the maleiimido groupreagents discussed above, an alkylation step may be used to causeconjugation. Conditions can be chosen such that, in the presence ofamino groups, essentially only thiol groups will be alkylated. With thisapproach, the reactions carried out can be represented typically byReaction 4 shown in FIG. 10 of the accompanying drawings. With thisapproach, the larger carrier molecule, for example flagellin, tetanustoxoid or the influenza subunit described herein, is first modified byreaction of a fraction of its amino groups with an active ester ofchloro, dichloro, bromo, or iodo acetic acid such as the compound ofFormula C shown in FIG. 9 of the accompanying drawings. This modifiedcarrier is then reacted with the sulfhydryl group in the polypeptide tobe modified, or a modified form of the polypeptide which has alreadybeen modified to contain a free thiol group (e.g. by thethiolactonization which is discussed above) if it did not originallyposses such a free thiol group. Such modification is described inExample XXV below. The reaction produces a thioether linkage byalkylation of the free-thiol (sulfhydryl) group.

It may be seen from an observation of the formulae of Structures (IV),(V), (IX), (X), (XI), (XII), (XIII), and (XIV) that a Cys amino acid,which in a reduced state provides an SH reactive group, is located ateither the C-terminal or N-terminal of the peptide structure. Thislocation permits the peptide to be chemically linked to carriermolecules at either terminus. Moreover, the Structures (XIV), (X), (IX),(X), (IV) have a six-proline spacer chain (Pro)₆ between the cysteineresidue and the remainder of the peptide sequence. This latterarrangement provides a chemical spacer between the coupled carrier andthe sequences representing a fragment of the natural hormone. Asix-proline spacer can be added as a side chain spacer, for example atposition 122 (lysine) in Structure (II), by initially adding an SH group(thiolactonization) to the free or unblocked epsilon amino group on this(lysine) residue, as set out in Example XXIX below. Then, utilizing theactivator A or B in FIG. 9 in which the component "X" is a chain of sixproline amino acids, conjugation can be carried out. In the latter case,a spacer is provided between the carrier and peptide linked at anintermediate site, for example at position 122 in Structure (II). In theformer case, only the spacer derived from the conjugating reagent linksthe carrier and peptide.

Modifying groups, such as hemocyanin from Keyhole limpet, containingfree amino groups can be prepared in buffer solution, such as phosphatebuffer, in sodium chloride solution at a pH of 6-8. To this solution,tolylene diisocyanate (T.D.I.C.) reagent diluted from about 1-10 toabout 1-40 times with dioxane is added to the modifying group. Thegeneral procedure was disclosed by Singer and Schick, J. Biophysical andBiochem. Cytology, 9, 519 (1961). The amount of T.D.I.C. added may rangefrom 0.075 to 1,000 molar equivalents of the modifier used. The reactionmay be carried out at about -5° to about +10° C., preferably 0° to 4°C., for about 1/2 to 2 hours. Any excess T.D.I.C. may be removed bycentrifugation. The precipitate may be washed with the above-mentionedphosphate buffer and the supernatants combined.

This activated modifying group solution may then be combined with thehormonal or non-hormonal polypeptide to be conjugated. The polypeptideis dissolved in the same phosphate buffer (5-30 mg/ml) and the volume ofmodifier and polypeptide combined according to the molar ratio of thetwo desired in the conjugate. Combined solutions are reacted at 300°-50°C., preferably 350°-40° C., for 3-6 hours.

Separation of modified polypeptide and free unconjugated polypeptide maybe accomplished by conventional techniques, such as gel filtration.

Picogram amounts of I¹²⁵ labeled polypeptide may be added as a tracer tothe reaction mixture at the time of conjugation, and a quantity ofpolypeptide conjugated to modifying groups (molar ratio) may bedetermined by the amount of radioactivity recovered.

Included in the methods for modifying the hormones, non-hormonalproteins and their fragments (unmodified polypeptides) are conjugationby use of water-soluble carbodiimide. The amino groups of the unmodifiedpolypeptide are first preferably protected by acetylation. This(acetylated) unmodified polypeptide is then conjugated to the modifier,such as a natural protein modifier, e.g. hemocyanin from Keyhole limpet,homologous serum albumin, and the like, or dextrans, Ficolls, orpolytyrosine, preferably in the presence of guanidine, such as guanidineHCl, using 10-ethyl-3-(3-dimethylaminopropyl)carbodiimide as activatingagent. This method is generally disclosed by Hoare and Koshland, Jr., J.of Biological Chemistry, 242, 2447 (1967). If Ficoll 70 is used, it ispreferred that it be first treated with ethylenediamine so as to renderthe final coupling more efficient. This treatment with ethylenediaminemay be performed in a solvent such as saline and dioxane at about roomtemperature and a pH of about 9-12, preferably 10-11, for about 1/4 toabout 2 hours. The conjugation itself between the unmodified polypeptideand the modifier may be performed in a solvent such as glycine methylester while maintaining the pH at about 4-5, preferably about 4.5-4.8.The temperature of reaction is conveniently about room temperature andthe reaction may be allowed to proceed for about 2-8 hours, preferably 5hours. The resulting modified polypeptide of this invention may bepurified by conventional techniques, such as column chromatography.

Modified polypeptides may also be prepared using glutaric dialdehyde asconjugating agent. According to a theory proposed by Richards andKnowles J. Mol. Biol., 37, 231 (1968)!, commercial glutaric dialdehydecontains virtually no free glutaric dialdehyde, but rather consists of avery complex mixture of polymers rich in alpha, beta-unsaturatedaldehydes. Upon reaction with natural protein modifiers such ashomologous serum albumins, these polymers form a stable bond through thefree amino group, leaving aldehyde groups free. This intermediateproduct then reacts with unmodified polypeptide in the presence ofalkali metal borohydride, such as sodium borohydride. This intermediateis formed at pH 7-10, preferably 8-9, at about room temperature. Themodified polypeptide is also conveniently obtained at about roomtemperature after about 1/4-2 hours reaction time. The resulting productis recovered in pure form by conventional techniques, such as gelfiltration, dialysis and lyophilization.

Polymerized sugar modifiers such as Ficoll 70 or Dextran T 70 may alsobe prepared for conjugation by treatment with a cyanuric halide, such ascyanuric chloride, to form a dihalotriazinyl adduct. The process may beperformed in a solvent such as dimethylformamide at about 0°-20° C.,preferably 10°-15° C., for about 1/2-4 hours. The resulting intermediateproduct may then be dialyzed until essentially halogen ion free, andlyophilized and treated with unmodified polypeptide at pH 8-11,preferably about 9-10, for about 1/2-12 hours at about 15°-35° C.,conveniently at room temperature. The resulting modified polypeptide maybe recovered as indicated above.

Said polymerized sugar modifiers may also be treated with an alkalimetal periodate, such as sodium periodate, at a pH of 3-6 at about30°-60° C. for about 1/2-4 hours, and the resulting intermediateconjugated with unmodified polypeptide at a pH of about 7-11, preferablyabout 8-10, for about 1/4 to about 2 hours at a temperature of about15°-80° C., preferably 20°-60° C. The resulting modified polypeptide ofthis invention may be separated as indicated previously.

The modifying groups may vary in chemistry and number for any givenpolypeptide structure. However, they will attach to only certain aminoacid moieties. In particular, when modifying with diazo groups, suchgroups will chemically bond to only the histidine, arginine, tyrosineand lysine moieties or sites. Other modifying groups will bond topeptide molecules at different sites and in different numbers.Consequently, depending upon the size and chemical make-up of theparticular modified polypeptide desired, one skilled in the art willreadily be able to calculate the maximum possible number of modifyinggroups associable with a polypeptide. It is also recognized that severalmodifying groups may attach themselves to each other which in turnattaches them to a single amino acid moiety, but as used herein,reference to a number of modifying groups means the number of reactionsites to which a modifier has been attached.

Throughout the foregoing description, the term "modified" or"conjugated" has been utilized in referring to the chemical reaction bywhich the foreign molecules become chemically attached to specific siteson the polypeptide. Although specific mechanisms by which this isaccomplished are described herein in detail, other appropriatemechanisms may be used if desired. It is clear that the modifier, i.e.,the substance which modifies the relevant polypeptide, can be aphysically larger molecule or fragment thereof than the molecule orfragment which it modifies. As noted above, such large molecules aredeemed herein to be "carriers". Clearly, physical size of the fragmentis not always critical, the criterion for effectiveness being that themammalian body's reaction generate antibodies in sufficient quanta andspecific to the targeted hormone or endogenous or non-endogenousprotein.

POLYMERIZATION

The instant modified polypeptides may also be prepared by polymerizationof the polypeptides from which they are derived, the term polymerizationbeing used herein to cover dimerization, trimerization, etc. Forexample, the modified polypeptides of the invention may be prepared bypolymerization of unmodified polypeptide using bi-functional imidoester.The imidoester, such as dimethyl adipimidate, dimethyl suberimidate anddiethyl malonimidate, may be used to form the polymer in a mannersimilar to the generally described methods of Hartman and Wold,Biochem., 6, 2439 (1967). The polymerization may take place convenientlyat room temperature in aqueous solvent at a pH of about 9-12, preferablyabout 10-11, over a period of 1/4-2 hours.

The instant modified polypeptides may also be prepared by dimerizationthrough a disulfide bond formed by oxidation of the thiol group on acysteine residue using iodosobenzoic acid and methods corresponding toknown methods, such as room temperature reaction for about 10-40minutes.

These relatively unsophisticated dimerization and polymerizationtechniques tend, however, to suffer from serious disadvantages.Dimerization of the polypeptide via a disulfide bridge has the advantageof not introducing any exogenous material into the animal (in contrastto the techniques discussed above which involve introduction ofexogenous carriers into the animal), but since the modified polypeptideadministered to the animal is only a dimer of the unmodified polypeptidewhich is not itself immunogenic to the animal, such dimers may in somecases be unsuccessful in provoking useful levels of antibodies.Polymerization using a bi-functional coupling reagent such as animidoester can provide a modified polypeptide large enough to bestrongly immunogenic. Unfortunately, experiments have proved thatstraightforward application of the bi-functional organic reagentpolymerization technique to either proteins or relatively largefragments thereof, which will often be required in practical use of thisinvention, produces very complicated mixtures of modified polypeptideshaving correspondingly complicated immunogenic properties. Furthermore,the immunogenic properties of the polymerized polypeptides thus producedare not readily reproducible, whereas such reproducibility is essentialin any material intended for pharmaceutical use, since the necessarytests of safety and efficiency cannot be performed on non-reproduciblematerial.

I have not found (though this knowledge is not disclosed in thepublished literature) that the reason for the very complicatedimmunogenic properties and the lack of reproducibility present in somepolymers produced by the bi-functional organic reagent polymerizationtechnique is that, notwithstanding the use of a bi-functional reagent,extensive cross-linking of the peptide tends to occur, suchcross-linking presumably being due to the presence of free amino, thiol,carboxyl and perhaps other groups (the exact groups involved dependingof course upon which groups the bi-functional organic reagent is capableof reacting with) at non-terminal positions on the polypeptide. Suchcross-linking produces branching and 3-dimensional structure in theresultant polymers. Not only does the relatively random cross-linkingthus produced render the structure of the polymers themselvesunpredictable and non-reproducible, but such cross-linking may wellalter the tertiary structure and shape of the unmodified polypeptidebeing polymerized, thus effecting its immunogenic properties (see theforegoing discussion of the importance of conformational determinants inthe antigenic properties of polypeptides and proteins).

There is a further, although usually minor, disadvantage which is sharedby both the bi-functional organic reagent polymerization technique andthe conjugation technique, namely the introduction of exogenousmaterials into the body of the animal being treated. The bi-functionalorganic reagent technique introduces a relatively small proportion ofexogenous material into the animal being treated (and even thisrelatively small proportion of non-endogenous material can be chosen sothat it is not strongly immunogenic), while the conjugation techniquetends to introduce a much higher proportion of non-endogenous materialand will usually provoke the formation of substantial quantities ofantibodies to the carrier as well as to the polypeptide. Although, asmentioned above, the formation of antibodies to the carrier (and in somecases to the bi-functional organic reagent used for coupling either inthe conjugation or polymerization techniques) may sometimes be useful(for example, a vaccine based upon an HCG peptide coupled to diphtheriatoxoid and intended for fertility control has the incidental advantageof also conferring protection against diphtheria), there are someoccasions on which it may not be desirable to provoke the formation ofrelatively large quantities of antibodies to the carrier; for example ifone wishes to use a vaccine containing a modified polypeptide of theinvention to treat a patient with a carcinoma or a serious viralinfection, it may be desirable to avoid overstraining the patient'simmune system by challenging it not only with the modified polypeptideto which antibodies are desired, but also with the carrier.

Accordingly, it is greatly preferred that, when producing the instantmodified polypeptides by the polymerization technique, thepolymerization be effected in such a way that coupling of the peptidefragments being polymerized occurs only at or near the terminals of thefragments, thus producing a true linear polymer substantially free ofnon-linear polymers of the fragments.

It may at first appear surprising that a linear polymer of apolypeptide, the monomeric form of which is effectively non-immunogenicto an animal, can be immunogenic to the same animal. It is believed(though the invention is in no way limited by this belief) that theincrease in immunogenicity upon polymerization is due to the increase inphysical size of the molecule, which enables the molecule to berecognized much more easily by the animal's immune system. It can beshown that at least some monomeric polypeptides are very weaklyimmunogenic and cause the animal's immune system to produce detectablequantities of antibodies, which quantities, however are much too smallto be effective. Immune systems are not well-adapted to recognizemolecules as small as the small polypeptides when the polypeptides arepresent in polymeric form.

Although the optimum number of polypeptide fragments in the modifiedpolypeptides will of course vary with the size of the individualfragments, the chemical nature of the fragments and perhaps the animalto which they are to be administered, in general I have found itconvenient to use polymers containing from 4 to 14 fragments. In mostcases, where it is desired only to affect a single hormone, it issimplest to use a polymer containing identical fragments, but it is notessential that all fragments of the polymer be identical and thefragments may be the same or different. For example, when it is desiredto produce a polymeric polypeptide for use in provoking antibodies toHCG, two or more of the polypeptides of Structures (I) to (XIV) abovecould be polymerized together so that the resulting polymer containedseveral different immunological determinants of HCG. Indeed, it is noteven necessary that all the polypeptides which are polymerized togethernecessarily be derived from the same protein; for example, if one wishedto influence a complicated hormonal system controlled by severaldifferent hormones, one might polymerize fragments of two or more of thehormones to form the polymer.

Polymerization of the fragments to form the linear polymericpolypeptides of the invention may be effected in any manner for couplingpeptide fragments to form linear polymers thereof known to those skilledin the art. The linear polymeric polypeptides of the invention may bedivided into two distinct types. In the first type, the individualpeptide fragments are linked head-to-tail by peptide linkages, so thatthe whole polymer comprises solely the fragments themselves and does notcontain any extraneous material. Although such pure polymers do have theadvantage of not introducing any extraneous material into the body ofthe animal being treated, they are usually too expensive to bepractical, since the necessary fragments (whether produced by totalsynthesis or cleavage of a natural protein) are themselves veryexpensive and substantial losses occur during the polymerizationprocess. Furthermore, the head-to-tail coupling of the fragments,without any intervening residues, may produce immunological determinantswhich have no counterpart in the unpolymerized fragment. For example, ifthe fragment described above, comprising the 105-145 sequence of HCG, ispolymerized by means of peptide linkages, a sequence:

    Pro-Ile-Leu-Pro-Gln-Asp-Pro-Leu-Thr

will be produced at each junction between adjacent fragments, and thissequence may provoke the formation of antibodies which would not beproduced by the fragment itself, and which may be undesirable. Incolloquial terms, since there is not "punctuation" to tell the immunesystem of the recipient animal where one fragment begins and anotherends, the animal's immune system may inadvertently start reading at thewrong residue and produce unwanted antibodies by running the sequencesof adjacent fragments together. For this reason, in general, I do notrecommend the use of linear polymers in which the fragments areconnected by peptide polymers, though of course such linear polymers maybe useful in certain instances.

Various methods of coupling polypeptide fragments via peptide bonds areknown to those skilled in the art. For example, one fragment to becoupled may have its C-terminal carboxyl group blocked (e.g. byesterification) and be reacted with the other fragment, which has itsN-terminal amino group blocked, but its carboxyl group activated bymeans of an activating agent. Obviously, blocking of non-terminal aminoand carboxyl groups may be necessary. Also, as well known to thoseskilled in this field, it may be advantageous to attach one end of thepolymer being produced to a support, such as polystyrene resin support,the polymer only being detached from the support after polymerization iscompleted.

In the second type of linear polymer polypeptide of the invention, thepolypeptide fragments are connected to one another by means of residuesderived from a bifunctional reagent used to effect polymerization of thefragments, so that the final linear polymer is an alternating linearpolymer of polypeptide fragments and coupling reagent residues. Althoughthis type of polymer necessarily introduces some extraneous materialinto the animal being treated, the proportion of extraneous material canbe made considerably lower than it would be if the fragments werecoupled to a large carrier, such as diphtheria toxoid. The couplingreagent, which is necessarily a bifunctional coupling reagent to producea true linear polymer, can be chosen so that the residues it leaves inthe polymer are not strongly immunogenic (so that they do not place thestrain on the immune system of the recipient animal that, for example, alarge carrier molecule such as diphtheria toxoid would) and the presenceof these residues in the polymer has the advantage of substantiallyeliminating false immunological determinants produced by conjunction ofthe head of one fragment with the tail of an adjacent fragment, asdiscussed above.

To ensure that a true linear polymer is produced during thepolymerization process, one terminal of a first polypeptide fragment isreacted with the bi-functional coupling reagent so that the couplingreagent reacts with a group present at or adjacent one terminal of thefragment; for example, the coupling reagent may react with a N-terminalamino group, a C-terminal carboxyl group or a free thiol group presenton a C-terminal cysteine. Obviously, the nature of the coupling reagentused determines what group on the peptide reacts. In order to avoid anycross-linking and to ensure a reproducible product, it is important thatonly one site on the first fragment be available for reaction with thecoupling reagent so that the coupling reagent can only attach to thefirst fragment at this one site. As those skilled in this field areaware, if it is desired to use a fragment containing more than one groupwhich could react with the coupling reagent, the excess sites may beblocked by attaching suitable protective groups thereto. The productformed by reaction of the first fragment with the coupling reagent isthen reacted with a second fragment (which may be the same as ordifferent from the first fragment) having a single site available toreact with the second reactive group of the bifunctional bicouplingreagent, thereby coupling the first and second fragments by a residuederived from the coupling reagent. Following any necessary purificationof this dimeric product, it is then reacted with a further portion of acoupling agent which may be the same or different reagent from that usedto effect the first coupling) thereby reacting the free terminal ofeither the first or second fragment with the coupling reagent.Naturally, it is important to ensure that only one site on the dimer isavailable for coupling to the coupling reagent, and as will be apparentto those skilled in the art, blocking or unblocking of potentialreactive groups on the dimeric polypeptide may be necessary. The productof the reaction of the dimeric polypeptide with the coupling reagent isthen reacted with a third fragment having only a single site availablefor reaction with the remaining reactive group of the coupling reagent,thereby producing a linear polymer containing three polypeptidefragments. Obviously, this process can be repeated until the desiredsize of linear polymer has been produced.

It will be apparent to those skilled in this field that the bifunctionalcoupling reagents used to prepare the linear polymeric polypeptides ofthe invention should be asymmetric i.e. they should have two functionalgroups which react with different groups on the fragments beingpolymerized, since, for example, if one attempted to react abifunctional bicoupling reagent having two functional groups, which bothreacted with amino groups, with a first fragment having a single aminogroup, at least some of the first fragment would be dimerized via aresidue derived from the bifunctional bicoupling reagent. Suchdimerization may in theory be avoided by using a very large excess ofthe coupling reagent, but in practice it is undesirable to run the riskof producing even a small proportion of dimer. Similarly, in laterstages of the polymerization process, it will be even more undesirableto use symmetric coupling reagents, thereby running the risk ofdimerizing the partially formed polymers already produced.

In the preferred process for producing the linear polymeric polypeptidesof the invention already described, the polymer chain is begun with afirst peptide having no unblocked thiol group and having an uriblockedamino group only at its N-terminal (peptides containing thiol groupsand/or amino groups other than at the N-terminal may of course be usedif all these thiol and amino groups are blocked with any conventionalblocking agent). This first peptide is then reacted with an amino groupactivating agent, a preferred activating agent for this purpose being6-maleimido caproic acyl N-hydroxy succinimide ester (MCS); reaction ofthe peptide with this reagent is optimally effected at a pH of 6.6). Theactivating agent reacts with the amino group at the N-terminal of thefirst peptide to form an activated form of the first peptide; in thecase of MCS, it is the ester portion of the reagent which reacts withthe N-terminal group of the peptide. It is normally then necessary toremove excess activating agent before continuing the preparativeprocess. Once the excess activating agent has been removed, theactivated first peptide is reacted with a second peptide having aC-terminal cysteine in a reduced state (i.e. having an unblockedfree-thiol group), thereby causing coupling of the N-terminal of theactivated first peptide to the C-terminal of the second peptide via anactivating agent residue. Desirably, the resultant dimer is purified asdescribed in more detail below. Next, the dimer is again reacted with anamino-group activating agent and then with a second portion of thesecond peptide or with a third peptide, thereby producing a trimer. Thisprocedure is repeated until the desired chain length has been achieved.

In order to secure reproducible responses from the immune systems oftreated animals, it is important that the linear polymeric potypeptidesof the invention be used in the form of pure polymers in which all themolecules contain the same number of fragments. To achieve such purepolymers, effective purification should be used after eachpolymerization step of the polymerization process. Because of the closechemical similarity between polymers containing different numbers offragments, chemical purification is ineffective, so purification must beeffected by physical methods. Gel filtration may be used if desired, butmy preferred purification method is reverse-phase, high-pressure liquidchromatography, preferably using a molecular sieve as the solid phase.

In this method of forming linear polymers, the first and second peptidesmay be identical in chemical configuration except that in the firstpeptide the C-terminal cysteine has a blocked thiol group.

As already mentioned, two particularly preferred fragments for use inthe linear polymeric polypeptides of the invention intended forprovoking antibodies to HCG are: ##STR6##

These first two preferred fragments for forming linear polymericpolypeptides of the invention to form antibodies to HCG mentioned abovemay be described as (111-145)-Cys and (105-145)-Cys, where the figuresrefer to the amino acid sequence in the beta subunit of HCG. It will beappreciated that, when these fragments are to be used in forming linearpolymeric polypeptides of the invention by the method just described,the lysine residue at position 122 must have its amino group blockedand, in the case of the (105-145)-Cys fragment, the non-terminalcysteine at position 110 must have its thiol group blocked, preferablywith an acetamidomethyl group.

It will be noted that some of the polymerization techniques discussedabove require the presence of a C-terminal cysteine on the peptide.Obviously, if it is desired to use a peptide which lacks a C-terminalcysteine as a second or later fragment in preparing the linear polymericpolypeptides of the invention by the preferred techniques discussedabove, it will be necessary to add a C-terminal cysteine to the peptide;appropriate methods for doing so are of course well known to thoseskilled in the field of polypeptide synthesis. Also, some peptides mayof course require blocking of non-terminal amino and/thiol groups beforeuse.

MISCELLANEOUS TECHNIQUES FOR MODIFYING POLYPEPTIDES

Numerous other techniques for the chemical modification of polypeptidesmay be employed in the practice of this invention. For example,naturally occurring proteins or polypeptides may be modified by removalof moieties therefrom. Some natural proteins have carbohydrate residues,especially sugar residues, attached to the protein chain and thesecarbohydrate residues may be removed according to methods known in theart, for instance by use of N-acetyl neuriminidase or N-acetylglucosidase, materials known to be used for removal of specificcarbohydrate residues.

Modification of the conformation of natural proteins by the breaking ofdisulfide bridges therein has already been referred to above inconnection with the choice of polypeptide to be modified in the instantinvention. However, it should be noted that in some cases breaking of anappropriate number of disulfide bridges within a protein may itselfcomprise a sufficient modification to render the protein much moreimmunogenic, and hence constitutes a sufficient chemical modification ofthe protein within the meaning of the instant invention. For example, asalready mentioned, the natural form of beta-HCG contains 12 cysteineresidues linked to form six disulfide bridges and any number of thesebridges may be broken using known techniques, as set out for example in:

Bahl, Biochem. Biophys. Res. Comm., 70, 525-532 (1976).

This particular article describes cleaving 3-5 of the six disulfidebridges in the beta subunit of HCG, but the same techniques may be usedto break all six bridges if so desired.

ADMINISTRATION OF THE INSTANT MODIFIED POLYPEPTIDES

Obviously, in order that the modified polypeptides of the invention canprovoke the formation of antibodies to the target protein within thebody of an animal, they must be administered to the animal in such a waythat they can come into contact with the cells responsible for formationof antibodies. In practice, this essentially means that the modifiedpolypeptides must be introduced into the circulatory system of themammal to which they are administered. Although the use of other modesof administration is not absolutely excluded, in view of the molecularsize and weight of most of the instant modified polypeptides likely tobe used in practice, the normal route or administration will beparenteral administration i.e. by injection. In the vast majority ofcases, the quantity of modified polypeptide which will need to beadministered will be far too small for convenient handling alone, and inany case the chemical nature of most of the modified polypeptidesprevents them being produced in a pure form free from liquid vehicles.Accordingly, it is normally necessary to administer the modifyingpolypeptides of the invention as a vaccine comprising a modifiedpolypeptide together with a vehicle. As already mentioned, a preferredvehicle for administration of the instant modified polypeptidescomprises a mixture of mannide monooleate with squalane and/or squalene.It has been found that this vehicle has the effect of increasing thequantity of antibodies provoked by the linear polymeric polypeptide,antigen or modified antigen of the invention when the vaccine isadministered to an animal. To further increase the quantity ofantibodies provoked by administration of the vaccine, it is advantageousto include in the vaccine an immunological adjuvant. The term "adjuvant"is used in its normal meaning to one skilled in the art of immunology,namely as meaning a substance which will elevate the total immuneresponse of the animal to which the vaccine is administered i.e. theadjuvant is a non-specific immunostimulator. Preferred adjuvants aremuramyl dipeptides, especially:

    NAc-nor Mur-L.Ala-D.isoGln

    NAc-Mur-(6-0-stearoyl)-L.Ala-D.isoGln or

    NGlycol-Mur-L.alphaAbu-D.isoGln

Thus, vaccines of this invention may be administered parenterally to theanimals to be protected, the usual modes of administration of thevaccine being intramuscular and sub-cutaneous injections. The quantityof vaccine to be employed will of course vary depending upon variousfactors, including the condition being treated and its severity.However, in general, unit doses of 0.1-50 mg. in large mammalsadministered from one to five times at intervals of 1 to 5 weeks providesatisfactory results. Primary immunization may also be followed by"booster" immunization at 1 to 12 month intervals.

To prepare the vaccines of the invention, it is convenient to first mixthe modified polypeptide, antigen or modified antigen of the inventionwith the muramyl dipeptide (or other adjuvant) and then to emulsify theresultant mixture in the mannide monooleate/squalene or squalanevehicle. Squalene is preferred to squalane for use in the vaccines ofthe invention, and preferably about 4 parts by volume of squalene and/orsqualane are used per part by volume of mannide monooleate.

As already noted, the modified polypeptides of this invention may beadministered parenterally to the animals to be protected, preferablywith a pharmaceutically acceptable injectable vehicle. They may beadministered in conventional vehicles with other standard adjuvants, asmay be desirable, in the form of injectable solutions or suspensions. Asindicated earlier, the adjuvant serves as a substance which will elevatetotal immune response in the course of the immunization procedure.Lipasomes have been suggested as suitable adjuvants. The insoluble saltsof aluminum, that is aluminum phosphate or aluminum hydroxide, have beenutilized as adjuvants in routine clinical applications in man. Bacterialendotoxins or endotoxoids have been used as adjuvants as well aspolynucleotides and polyelectrolytes and water soluble adjuvants such asmuramyl dipeptides. The adjuvants developed by Freund have long beenknown by investigators; however, the use thereof is limited to non-humanexperimental procedures by virtue of a variety of side effects evoked.The usual modes of administration of the entire vaccine areintramuscular and subcutaneous.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE I

This example illustrates the preparation of modified polypeptides of theinvention based upon primate reproductive hormones and the use of suchmodified polypeptides in altering the level of reproductive hormones inbaboons.

Adult female baboons were studied for at least one menstrual cycle forpatterns of urinary estrogens, plasma, progestin, and in some casesurinary LH. Only those animals displaying normal patterns of thesehormones were immunized. The criteria for normality and the proceduresfor housing animals are well known and will not be described.

GONADOTROPIN PREPARATIONS

Human Luteinizing Hormone (HLH)-partially purified preparation fromhuman pituitaries with a biological potency of 2.5 units per mg.(NIH-LH-SI).

Human Follicle Stimulating Hormone (HFSH)-a partially purifiedpreparation from human pituitaries with a biological potency of 86 unitsper mg. (NIH-FSH-SI).

Human Chorionic Gonadotropin (HCG)-a highly purified preparation fromhuman pregnancy urine with biological potency of 13,200 IU/mg. (2ndIRP-HCG).

Monkey Luteinizing Hormone (MLH)-a crude preparation from rhesus monkeypituitaries with a biological potency of 0.75 units per mg. (NIH-LH-SI).

Ovine Luteinizing Hormone (OLH) (NIH-LH-S5).

Baboon Luteinizing Hormone (BLH)-partially purified baboon pituitarypreparation with a biological potency of 1.1 units per mg. (NIH-LH-S1).

All preparations, excepting the OLH, were prepared in the inventor'slaboratory. LH and HCG biological activity was determined by the ovarianascorbic acid depletion test and the FSH preparation assayed by theovarian augmentation assay.

The hormones were chemically modified by coupling with a hapten invarying ratios of hapten to hormone as described by Cinader et al.,supra. For convenience, the Cinader process is discussed herein althoughPhillips, supra, may provide a more stable bond under certaincircumstances. In this procedure, the protein hormone serves as acarrier and the hapten is coupled to it by diazo bonds. Although avariety of hapten groups were coupled to different hormones, the samebasic procedure was used for any combination. Fifteen to thirty-fivehaptenic groups per hormone molecule were found most useful forpreparing immunizing antigens. The basic reaction consisted ofdiazotizing the hapten (sulfanilic acid) by adding it to a solution of0.11N.HCl and then slowly adding this solution dropwise to a 1 percentsolution of NaNO₂ with constant stirring at 4° C. Diazotization wasconsidered complete when free HNO₂ was detected in the reaction mixture.Although the above reaction was accomplished at 4° C., optimumtemperatures of the reaction normally are about 0°-6° C., although 4° C.is preferred.

The hapten-protein coupling was performed by dissolving the proteinhormone in an alkaline buffer, pH 8.0. The diazotized hapten was addedslowly to the hormone solution with continuous stirring at 4° C. The pHof the reaction was constantly monitored and kept near 8.0. After allthe hapten had been added, the pH was finally adjusted to 8.0, and thereaction mixture was stirred for 1-2 hours and allowed to stand at 4°overnight. The mixture was thoroughly dialyzed for 6-8 days againstdistilled water to remove unreacted hapten.

Although the number of diazo groups per hormone molecule could beregulated by the number of moles of hapten and hormone reacted, aparallel control experiment, with S³⁵ labelled sulfanilic acid toevaluate the precise composition of the hapten-protein samples, wasperformed with each diazotization. The same hormone preparation to beused for immunization was used in the control experiment. After thereaction was completed, an aliquot was taken from the reaction mixtureand the remainder thoroughly dialyzed. Equal volumes of the dialyzed andundialyzed solutions were counted by liquid scintillation. By comparingthe counts of the dialyzed and undialyzed samples, the moles of haptencoupled to each mole of hormone were calculated since the unreactedhapten was removed by dialysis. For this calculation, a molecular weightof 30,000 was assumed for all gonadotropin preparations.

Following dialysis, hapten-hormones were lyophilized and stored at 4° C.Diazo-HCG (35 groups/molecule) and HLH (26 groups/molecule) werebioassayed by the ovarian ascorbic acid depletion method and found toretain 62 and 85 percent respectively of the activity of the unalteredhormones from which they were derived. None of the other hormones wasassayed for biological activity.

IMMUNIZATION PROCEDURES

Female baboons received their initial immunization on days 3-5 of themenstrual cycle and the second and third injections one week apart. Thefourth injection was given 2-3 weeks after the third. A few animalsreceived a fifth injection at 70-80 days after the first injections. Allantigens were administered subcutaneously in a suspension of mannidemonooleate or peanut oil. Doses of antigens for each injection variedbetween 3 and 5 mg. Injection sites were inspected daily for 5 daysafter each immunization for local reactions.

MONITORING EFFECTS OF IMMUNIZATION

Daily 24-hour urine specimens and frequent serum samples were collectedduring at least one menstrual cycle prior to immunizations and followingimmunizations until the effects of treatment were assessed. Urinary LH,urinary estrogens and plasma progestins were measured. Antibodies weredetected in post-immunization serum samples by reacting 0.2 ml. of a1:1000 dilution of serum in phosphate-buffered saline (pH 7.4) 0.5percent normal baboon serum with 250 pg of 1¹³¹ labelled hormone. Serawere reacted with both the unaltered immunizing hormone and unalteredbaboon LH for antibody detection. A purified baboon LH preparation(1.9×NIH-LH-S1) was used as a tracer antigen. Antigen-antibody complexeswere precipitated with ovine anti-baboon gamma globulin after a 24-hr.incubation at 4° C. Antibody levels were expressed as pg of labelledhormone bound. Significant antibody levels were considered to be thosethat would bind 5.0 pg or more of the 1¹³¹ labelled antigen.

Antisera were fractionated by gel filtration on Sephadex G-200 accordingto the procedure of Fahey and Terry (at p. 36, Experimental Immunology,F. A. Davis Co., Philadelphia, Pa., 1967, incorporated by reference tothe extent necessary to understand the invention) to determine theproportion of IgM and IgG antibodies in the baboon sera. Since the IgGfraction in this procedure contained a portion of IgA and IgDantibodies, only and total titers were determined. The IgM fraction fromthe column was reacted with I¹³¹ hormones and the binding capacitydetermined. The volumes of the fractionated sera were adjusted so thatantibody levels would be comparable to those of whole serum.

ANTIBODY PRODUCTION

No significant reactions were observed at the site of injectionfollowing any immunization. On 4 occasions, a slight induration (2-3 cmin diameter) was seen when mannide monooleate was used as a vehicle butthe redness and swelling disappeared within 4-5 days. Antibodies weredetected against the immunizing antigen within 3-5 weeks in all animals.The extent, duration and cross reactivity of these antibodies isrecorded. Generally speaking, higher levels were observed toheterologous gonadotropin immunization than to homologous ones.

The cross-reactivity of induced antibodies with baboon LH was studied oneach animal. Cross-reactivity of antisera at peak levels was recorded.Although relatively high antibody activity against human LH and HCG wasseen, relatively little reaction with baboon LH occurred. Anintermediate cross-reaction was noted with anti-ovine LH and a highdegree of cross-reactivity was seen with anti-monkey LH. Diazo-human FSHwas weakly antigenic in the baboon. The duration of antibody productionwas generally longer with the human and sheep gonadotropin immunizationthan with those of monkey or baboon origin.

Peak antibody levels usually occurred at the time when the antibodieshad shifted to principally the IgG type. Early antibodies had a largerproportion of IgM type and were generally more cross-reactive withbaboon LH. The change in the proportion of the total antibody populationwith IgM was recorded from the time antibodies were first detected.Significant cross-reactivity to baboon LH was observed in anti-humangonadotropins when IgM was abundant but dropped sharply as the antiserashifted to nearly all IgG. This drop in cross-reactivity did not occurwith monkey and baboon immunizations. Again, the ovine LH immunizationsproduced an intermediate change in reactivity with the shift from toIgG.

EFFECTS ON THE MENSTRUAL CYCLE

The effects of immunization upon the event of the menstrual cycle weredetermined by observing changes in sex skin turgescence and levels ofpituitary and/or ovarian hormones. Based on these parameters, the delayor retardation of ovulation from the expected time, as judged by thecontrol cycle, was calculated. One animal immunized with ECG had nointerruption in ovulation and another immunized with HFSH was delayedfor only one cycle. Two animals injected with HLH and two injected withECG had ovulation delays equivalent to two menstrual cycles. A thirdanimal immunized with HLH was delayed a calculated 86 days. Ovine LHimmunizations produced an 88 day delay in ovulation.

Immunizations with diazo-monkey or baboon LH resulted in longerdisruption of the menstrual cycle. Calculated delays in ovulation forthe two animals receiving monkey LH were 146 and 122 days, whereas theanimals receiving altered baboon LH were retarded from ovulation 224 and210 days.

Effects on specific hormone patterns following immunization with HLH inone animal were recorded. The interval between menses was considered torepresent a "cycle". Urinary estrogens and plasma progestin patternsindicated that no ovulation occurred during the cycle of immunizationwhich was 85 days in duration. Urinary estrogens were elevated duringtreatment but did not reflect a typical pattern. Plasma progestin werenot elevated until about day 19 of the first post-treatment cycle.Antibody levels were elevated from about day 35 of the treatment cycleuntil 289 days from the first detection of antibodies. An LH assay wasnot available when this animal was studied and no data on plasma orurinary levels of this hormone were obtained.

Hormonal patterns following an immunization with diazo-baboon LH wererecorded. In this animal, antibody levels were lower and persisted, ingeneral, for a shorter period than did immunizations with humangonadotropins. During the treatment cycle, levels of urinary estrogensand plasma progestins followed a normal pattern but were quantitativelylower than normal. Urinary LH patters fluctuated markedly due to theinjections of diazo-LH during this period. No conclusive evidence ofovulation was obtained for the treatment cycle. The first post-treatmentcycle lasted 246 days. During this cycle urinary LH and estrogens wereelevated on days 35-41 but there was no subsequent elevation in plasmaprogestins that would indicate ovulation had occurred. Following day 42of this cycle, there was no significant elevation in any of the threehormone levels until day 231 when significant elevations of urinaryestrogens and LH occurred. These rises were followed 3 days later by anelevation in plasma progestins indicating the presence of a functioningcorpus luteum. A second post-treatment menstrual cycle was of normalduration and the endocrine patterns were normal.

Antibodies to unaltered baboon LH attained maximum levels by about day70 of the post-treatment cycle and remained relatively constant untilday 190 when a steady decline was observed. By day 215 of this cycle,antibody levels were barely detectable. Approximately 16 days after thistime, a peak of LH commensurate with a normal midcycle elevation wasobserved. From this point the animals appeared to have the normalfunction of the pituitary-ovarian axis. Hormonal patterns in animalswith other heterologous gonadotropin immunizations were similar to theanimal receiving HLH and other animals receiving monkey or baboon LHwere similar in response to the animal receiving baboon LH.

These results in baboons indicated that the modification of areproductive hormone, by the procedures outlined, did render itantigenic and the antibodies thus formed did neutralize naturalendogenous hormones if the natural hormone was obtained from the speciesreceiving the immunizations with modified hormone.

EXAMPLE II

This example illustrates the preparation of a modified polypeptide ofthe invention derived from HCG and its effect on the levels ofreproductive hormones in a human female.

HCG is a hormone naturally present only in pregnant women with theexception that an entity at least analogous thereto has been found to bepresent in humans in conjection with neoplasms. HCG is also commerciallyavailable. Human LH is immunologically and biologically identical to HCGeven though there are chemical differences. Since they are biologicallyidentical and HCG is readily available from commercial sources it waspresumed that the effectiveness of this immunological procedure could beevaluated by injecting modified HCG into nonpregnant women andmonitoring the blood levels of LH. Antibodies formed will neutralizeboth the LH and the modified HCG. Reference in the above regard is madeto the publications identified earlier herein.

Women have a pattern of LH levels; the level is substantially constantuntil the middle period between menstrual cycles, immediately prior toovulation; at that point the LH level rises greatly and helps induce theovulation. Monitoring the LH level and the antibody level will show thatthe procedure used did or did not cause the production of antibodiescapable of neutralizing the endogenous reproductive hormone, namely LH.

A woman aged 27 years was selected for study. Hormone was obtained,purified and modified as described in more detail below. The modifiedhuman hormone (HCG) was injected into the subject. It is well known thatantibodies to HCG react identically to LH as well as HCG. The effect ofthe immunization was evaluated, principally by monitoring blood levelsof LH. Finally the results were evaluated.

PREPARATION OF HORMONE

Clinical grade HCG derived from pregnancy urine was obtained from theVitamerican Corp., Little Falls, N.J. This material has an immunologicalpotency of 2600 IU/mg. Contaminants were detected in this preparation.Purification consisted of chromatography and elution. Fractions weredialyzed and lyophylized. The most potent fraction containedapproximately 7600 IU/mg.; however, it was heterogeneous onpolyacrylamide gel electrophoresis.

The fraction was further purified by gel filtration. The elution profilerevealed two major protein peaks. The most potent HCG was found in thefirst peak and had an immunological potency of 13,670 IU per mg. Thisfraction was subjected to polyacrylamide gel electrophoresis. Furtherpurification by gel filtration showed no evidence of heterogeneity ofthe HCG at this stage. Consequently, materials for study were processedaccording to the above procedure.

The contamination of this purified HCG was tested with I¹³¹ used foridentification and a sample was reacted with antisera against severalproteins offering potential contamination. Those proteins were folliclestimulating hormone, human growth hormone, whole human serum, humanalbumin, transferrin, alpha one globulin, alpha two globulin andorosomucoid. No detectable binding of the purified HCG was observed withany antisera at a dilution of 1:50 of each. These negative results,calculated against potential binding of the respective proteins,indicated that contamination if any was less than 0.005 percent.

ALTERATION OF HORMONE

Hormone was altered by coupling with a hapten (sulfanilazo). This methodcouples the hapten molecules to the protein via the amino group of thealiphatic or aromatic portion of the hapten. The number of haptenmolecules coupled to each HCG molecule (Ha-HCG) could be regulated andfor this study, forty haptenic groups per HCG molecule were used forpreparing the immunizing antigen.

Following the hapten-coupling process, the Ha-HCG was sterilized andtested.

SUBJECT

The subject was multiparous and had terminated her reproductivecapabilities by prior elective bilateral salpingectomy. She was in goodhealth and had regular cyclic menstruation. She underwent completehistory, physical examination and laboratory evaluation including bloodcount, urinalysis latex fixation and Papanicolaou smear. She had nohistory of allergy.

To demonstrate normal functioning of the pituitary-ovarian axis prior toimmunization, blood samples were obtained every other day from the firstday of menses for 10 days, then daily for 10 days and finally, everyother day until the next menses. Serum determinations of FSH, LHestrone, estradiol and progesterone were performed. These studiesindicated an ovulatory pattern.

IMMUNIZATION PROCEDURES

Ten mg. of the Ha-HCG antigen were dissolved in 1.0 ml. of saline andemulsified with an equal volume of oil. Prior to injection, scratchtests to antigen and vehicle were performed. Immunizations were begun inthe luteal phase of the treatment cycle to prevent superovulation fromthe administered HCG. Four injections at two week intervals were givento the subject. The first two of these were administered in oilsubcutaneously (1.0 ml in each upper arm) the final two injections weregiven in saline only via the intradermal route. Following eachinjection, blood pressure readings were taken and the subject observedfor allergic reactions.

MONITORING EFFECTS OF IMMUNIZATIONS

Blood samples were collected at weekly intervals beginning two weeksafter the initial injection to test for the presence of humoral andcellular antibodies. Following completion of the immunization schedule,blood samples were collected in the same manner as in the control cycleto assess effects of immunization on hormonal patterns of the menstrualcycle. Since antibodies to HCG react identically to LH as with HCG, LHwas monitored as an index of effectiveness of the procedure. A thirdcycle was similarly studied six months after initial immunization. Uponcompletion of the study, physical and pelvic examinations and laboratoryevaluations were repeated.

Serum samples from the control and post-treatment cycles were assayedfor FSH, LH, estrone, estradiol and progesterone.

The subject was tested for delayed hypersensivity before immunizationand at two week intervals until the injection schedule was completed byan in vitro lymphocyte transformation test.

RESULTS

Temporal relationships of serum pituitary and gonadal hormones in thecontrol cycles of the subject were recorded. Antibody titers to HCG weredetected in the subject after two injections. Menses occurred at regularintervals during the immunizations.

Following the initial injection in mannide monooleate, some itching andswelling at the injection site occurred. Subsequent intradermalinjections in saline produced no reactions and it was concluded that thelocal reactions were induced by the mannide monooleate. Lymphocytetransformation tests on plasma samples were negative.

In the post-treatment cycle, baseline follicular and luteal phase LHlevels were not noticeably changed in the subject. Very small midcycleelevations in LH levels were observed as compared to the normal largeincreases. FSH patterns in the post-treatment cycle were normal. Thisindicated that the antibodies were neutralizing the action of endogenousLH.

The subject showed no ovulatory progesterone pattern but attainedrelatively high antibody titers to LH and HCG after only two injectionsof Ha-HCG.

The subject was studied during another cycle approximately six monthsfrom the first immunization. Significant antibody titers were found. LHpatterns indicated a small midcycle elevation. FSH patterns wereessentially normal. Thus, the specificity of anti-HCG antibodies to LHwas shown but not to FSH.

The following Examples III-VII illustrate further experimental resultsobtained by administration of the vaccine prepared in Example II above.

EXAMPLE III

Another woman aged 29 years was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to the results found in Example II except that(1) the levels of estrone and estradiol were substantially normal, (2)the subject acquired significant antibody titers late in thepost-immunization cycle, and (3) in the cycle studies after six monthsthis subject showed no significant midcycle elevation in LH patterns.

EXAMPLE IV

Another woman aged 29 years was selected for further study. Hormone wasobtained and purified and modified as in Example II. This modifiedhormone was injected into this subject in the same way as in Example II.The subject was monitored and tested as in Example II.

The results were similar to the results found in Example II except that(1) baseline follicular and luteal phase LH levels were noticeablydepressed in the post-treatment cycle, (2) no midcycle elevations wereobserved in LH, (3) estrone levels were elevated during the follicularphase of the post-immunization cycle, and (4) during the six-monthsstudy there was no significant midcycle elevation in LH patterns.

EXAMPLE V

Another woman aged 35 years was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to the results found in Example II except that(1) baseline follicular and luteal phase LH levels were noticeablydepressed in the post-treatment cycle, (2) a very small midcycleelevation of LH was observed, (3) levels of FSH patterns in thepost-treatment cycle were depressed, and (4) levels of both estrone andestradiol were reduced during the follicular phase of thepost-immunization.

EXAMPLE VI

Another woman aged 28 years was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to results found in Example II except that (1)baseline follicular and luteal phase LH levels were depressed in thepost-treatment cycle, (2) no peaks were observed in the midcycle levelsof LH, (3) estrone levels appeared elevated in the follicular phase ofthe post immunization cycle, and (4) LH patterns indicated nosignificant midcycle elevation in the six-month post-immunization cycle.

EXAMPLE VII

Another woman aged 28 years was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to results found in Example II except that (1)antibody titers to HCG were not detected until after three injections,(2) baseline follicular and luteal phase LH levels were depressed in thepost-treatment cycle, (3) no peaks nor midcycle elevation in the LH wereobserved, (4) estrone levels were elevated during the follicular phase,and (5) no significant antibody titers were found in the six monthcycle.

All the above examples show the practicality of injecting modifiedhormones for the purpose of neutralizing an endogenous reproductivehormone and thereby offering a procedure for the prevention ofconception or the disruption of gestation.

EXAMPLE VIII

Data obtained in earlier experiments and discussed in Examples I-VIIshowed that a modified natural reproductive hormone, when injected intoan animal of species from which it was derived, would produce antibodiesthat would neutralize the action of the unmodified endogenous naturalhormone in the body of the animal. Mormones used in Examples I-VII wereFSH, LH and HCG. New experiments were performed, based on thisknowledge, to identify another reproductive hormone (placental lactogen)that could be used in a similar fashion.

PREPARATION OF HORMONE

A purified preparation of placental lactogen was prepared from placentaeof baboons since it was intended to use modified placental lactogen toimmunize baboons. Placentae were extracted and purified by columnchromatography according to previously published procedures. The puritywas tested by polyacrylamide gel electrophoresis and byradioimmunoassay. The material obtained showed a high degree of purityon electrophoresis, and radioimmunoassay showed no contamination withother placental hormones.

HORMONE MODIFICATION AND IMMUNIZATIONS

The baboon placental lactogen (BPL) was altered by coupling with thediazonium salt of sulfanilic acid as outlined for other hormones inExample I. The number of diazo molecules per BPL molecule in thisinstance was 15. Immunization procedures were similar to those describedin Example I for other hormones.

RESULTS

Within 4-6 weeks after the first injection of diazo-BPL, antibody levelsto natural unmodified BPL in vitro were detected in 6 female baboons.Levels rose to a plateau within 8-10 weeks and remained there forseveral months. Hormonal measurements indicated that there were noeffects on the normal events of the menstrual cycle due to theimmunizations. Since BPL is normally secreted only in pregnancy, thiswas not a surprising observation.

All six females were mated with a male of proven fertility three times(once in each of three different cycles during the fertile period).Pregnancy diagnosis by hormonal measurement was performed after eachmating. From the 18 matings, there were 13 conceptions as judged bypregnancy tests. The animals that were pregnant had menstrual bleeding7-12 days later than was expected for their normal menstrual cycles.Subsequent hormonal measurements confirmed that these 13 pregnancieswere terminated by abortions approximately one week after the time ofexpected menses.

The findings suggest that the antibodies formed in the animal's bodyafter immunization had no effect on the nonpregnant menstrual cycle butwhen pregnancy was established, they neutralized the baboon placentallactogen in the baboon placenta and the result was abortion very earlyafter conception.

When in Examples I-VIII above Structures (I), (II) and (III) aremodified by use of a diazosulfanilic acid, dinitrophenol, or S-acetomercaptosuccinic anhydride or structures (II) and (III) are modified byaddition of polytyrosine or polyalanine, according to known methods, theresults obtained should be similar to those in said Examples.

Similarly, when FSH, somatomedian, growth hormone or angiotension II aremodified by use of diazosulfanilic acid or trinitrophenol, the resultsobtainable upon administration of the purified modified polypeptide intoa male or female human or animal would indicate the stimulation ofantibodies which neutralize all or some of the modified polypeptide aswell as corresponding endogenous polypeptide.

EXAMPLE IX

This example illustrates the modification of levels of reproductivehormones in baboons following administration of modified polypeptides ofthe invention similar to those used in Example I above.

The subjects used in the studies reported in the example are femalebaboons. All baboons were adults of reproductive age. A description ofsubjects and the conditions of experimentation has been given in ExampleI. The animals were being studied using highly purified beta subunits ofHCG using a preparation with a biological activity of less than 1.0IU/mg. Animals were immunized with 14-26 moles/mole of polypeptide ofdiazosulfanilic acid coupled subunit in mannide monooleate.

Antibody levels were assessed by determining the binding of serumdilutions with I¹²⁵ labelled antigens. Cross-reactivity of antisera wasmeasured by direct binding of labelled antigens and by displacementradioimmunoassays. Antifertility effects in actively immunized animalswere tested by mating females with males of proven fertility. Effects inpregnant baboons passively immunized with either sheep or baboonanti-beta-HCG were determined by monitoring serum levels ofgonadotropins and sex steroid hormones before and after immunizations.

Eight female baboons were immunized with the modified beta subunit ofHCG. Significant antibody levels were attained in all animals.

Baboon immunizations with the modified beta subunit of HCG resulted inhigh antibody levels reacting to HCG, human LH and baboon CG but not tobaboon LH. All animals remained ovulatory; however, no pregnanciesresulted from numerous matings with males of proven fertility. Passiveimmunization of non-immunized pregnant baboons with sheep anti-beta-HCGserum produced abortions within 36-44 hours.

The following Examples X-XVI illustrate chemical modification techniquesused to produce the modified polypeptides of the invention.

EXAMPLE X

Hemocyanin from Keyhole limpet (KLH) solution (7 mg/ml) in 0.005M sodiumphosphate buffer 0.2M in NaCL, and of pH 7.5, is prepared. Insolubleparticles are removed by centrifugation. To one ml. of this solution,tolylene diisocyanate (T.D.I.C.) reagent is added (20 microl.) dilutedto 1/30 with dioxane, the amount being essentially the equivalent of themoles of lysyl residues in the KLH molecules. After 40 minutes at 0° C.,the T.D.I.C. activated KLH solution is combined with 0.5 mg of syntheticbeta-HCG peptide having the following structure: ##STR7## which is firstdissolved in 25 microl of 0.05M sodium phosphate buffer 0.2M in NaCl,and of pH 7.5. The mixture is incubated at 37° C. for four hours. Theresulting product is purified by gel filtration.

EXAMPLE XI

One g. of Ficoll 70 is dissolved in 1 ml each of normal saline and 2Methylenediamine (adjusted to pH 10 with hydrochloric acid) solution. Thesolution is kept at room temperature in a water bath and stirred with amagnetic stirrer. Cyanogen bromide, 4 g, dissolved in 8 ml of dioxane,is added to the Ficoll 70 solution. The acidity of the mixture ismaintained at pH 10-10.5 for 8 minutes by adding drops of 2N sodiumhydroxide solution. An additional 2 ml of 2M ethylene diamine, pH 10,solution is added, and stirring at room temperature is continued for 30more minutes. The product is purified by passing it through a Bio-Gelp-60 column.

EXAMPLE XII

Two mg of the compound of Structure (II) containing a picogram amount ofI¹²⁵ labeled adduct and KLH (1.6 mg) is dissolved in 1 ml. of 1.0Mglycine methyl ester in 5M guanidinehydrochloride. 19.1 mg. of ethyldimethylamino propylcarbodiimide (E.D.C.) is added to this solution. Theacidity is adjusted to and maintained at pH 4.75 with 1N HCl at roomtemperature for 5 hours. The KLH-peptide conjugate is purified bypassing it through a Bio-Gel p-60 2.2×28 cm column equilibrated with0.2M NaCl.

EXAMPLE XIII

Solid bifunctional imidoester dihydrochloride (3 mole) is added in 2 mgportions at 5-minute intervals to a constantly stirred solution of 1mole of polypeptide of Structure (II) (1-20 mg/ml) in 0.1M sodiumphosphate, pH 10.5 at room temperature. 0.1N Sodium hydroxide, is addedto maintain the acidity at pH 10.5. One hour after the addition of thediimidoester has been completed, a polymerized product according to thisinvention is obtained.

EXAMPLE XIV

To a 20 mg/ml solution of homologous serum albumin in 0.1M boratebuffer, pH 8.5, 1000% mole excess of 25% aqueous solution of glutaricdialdehyde is added at room temperature. The excess dialdehyde isremoved by gel filtration in water using Bio-Gel p-2. The materialcollected at the void volume is lyophilized, and the dried product isredissolved in 0.1M borate buffer, pH 8.5 (20 mg/ml), mixed with therequired amount of polypeptide of the following Structure: ##STR8## 20mg/ml) in the same buffer at room temperature. Twenty minutes later,sodium borohydride in 250 percent molar excess of polypeptide XVI isadded. The reaction is terminated after one hour. The conjugated productis purified by gel filtration on, Bio-Gel p-60 column, dialyzed free ofsalt and lyophilized.

EXAMPLE XV

1 g of Ficoll 70, 500 mg of NaHCO₃, 3 g of cyanurin chloride, 20 ml ofH₂ O, and 80 ml of dimethylformamide, are stirred at a temperature below16° C. for 22 hours. The product is dialyzed against distilled wateruntil Cl-free, then lyophilized. 2 mg of the polypeptide of Structure(XV) containing a minute quantity of I¹²⁵ labeled analogue is incubatedwith 1 mg of this product in 0.25 ml of 0.2M. sodium borate buffer, pH9.5, for one hour at 20° C., and the product is recovered from a Bio-Gelp-60 2.2×28 cm column.

When the above procedure is carried out and Dextran T 70 is used inplace of Ficoll 70, the corresponding modified polypeptide, usefulaccording to this disclosure, is obtained.

EXAMPLE XVI

Ficoll 70, 1.2 g of NaIO₄, and 0.42 g of KCl are dissolved in 1.5 ml of1M sodium acetate buffer, pH 4.5, and incubated at 37° C. for 1 hour.

Two mg (=588 micro moles) of polypeptide of Structure (XV) containing aminute quantity of I¹²⁵ labeled analogue is incubated with 2 mg of theproduct obtained above in 0.3 ml of 0.2M borate buffer, pH 9.5 at 55° C.for 1 hour. The reaction mixture is then chilled in an ice-water bathand 1 mg of NaBH₄ 1 mg is then added into this solution. The reductionreaction is terminated by passing the product through the Bio-Gel p-602.2×28 cm column equilibrated and eluted with 0.2M NaCl.

EXAMPLE XVII

Numerous rabbits are immunized with a variety of synthetic peptidesconjugated to different modifying groups. Following two or threeimmunizations at 3-5 week intervals, sera from animals are assessed bydetermining their ability to bind in vitro to radiolabeled MCG. Thespecificity of this binding is studied by reacting the same sera againstother similarly labeled protein hormones, particularly pituitary LH.Sera are further assessed by determining their ability to inhibit thebiological action of exogenously administered HCG in bioassay animals.Thus, the increase in uterine weight of the immature female rat inresponse to a prescribed dose of HCG is noted. The dose of HCG isadministered subcutaneously in saline in five injections over a threeday period and the animal is sacrificed for removal of the uterus on thefourth day. The weight of the uterus increases in dose responsivefashion with the hormone injections. When assessing the effects ofantisera in this response, varying quantities of test serum areadministered intraperitoneal separately from the subcutaneous injectionof hormone during the assay. This procedure permits the antiserum to beabsorbed rapidly into the rat's bloodstream and will permit interactionof it with hormone when the latter likewise enters this fluid. If theantiserum is capable of reacting with the hormone in a manner preventingstimulation of the uterus, the antiserum is considered to be effectivefor biological inhibition of hormone action.

The frequency of animals showing a positive response to immunologicalbinding and neutralization of biological activity is documented invarious of my publications.

EXAMPLE XVIII

Iodosobenzoic acid (dissolved in a slight excess of 1N potassiumhydroxide) in 10% molar excess is added to the peptide of Structure (II)in phosphate buffer with normal saline at pH of 7.0. After thirtyminutes at room temperature, the dimeric product polypeptide is purifiedby gel filtration.

EXAMPLE XIX

To an ice water bath cooled and vigorously stirred 0.23 ml. of bovinegamma globulin (10 mg/ml) in 0.05M phosphate buffer with normal saline(PBS) pH 7.5, 50 microl. of 1/10 T.D.I.C. in dioxane is added. After 40minutes, the excess T.D.I.C. is removed by centrifugation (0° C., 10minutes, 10,000 g) and the precipitate is washed twice with 0.1 ml ofPBS. The combined supernatants are added to 7.7 mg of the peptide ofStructure (II) dissolved in 0.8 ml. of PBS, pH 7.5. The mixture isstirred at room temperature for 10 minutes, then incubated at 37° C. for4 hours. The conjugate product is purified by dialysis.

EXAMPLE XX

BSA (10 mg/ml) in PBS solution (0.25 ml.) is treated with 50 microl. of1/10 T.D.I.C. dioxane solution and conjugated to 7.5 mg. of thesynthetic beta-HCG peptide of Structure (III) in 0.8 ml. of PBS (pH 7.5)as in Example XIX to obtain the product.

EXAMPLE XXI

To an ice water bath cooled and vigorously stirred 0.6 ml. of beta-HCGpeptide of Structure (III) (10 mg/ml) in phosphate buffered saline, pH7.5, is added 30 microl. of 1/10 T.D.I.C. dioxane solution. After 40minutes, the excess T.D.I.C. is removed by centrifugation (10,000 g, 0°C., 10 minutes) and the precipitate is washed twice with 0.1 ml. PBS.The combined supernatants are added to 3 mg of poly-(D, L-Lys-Als)dissolved in 0.3 ml. of PBS. The mixture is incubated at 37° C. for 4hours. The product is then dialyzed and lyophilized.

EXAMPLE XXII

The results set out in Table I provide further evidence of the broadapplicability of this invention as indicated previously in thisspecification.

Using standard methods of testing in rabbits, both immunological bindingresponse and neutralization of biological activity were established forthe modified polypeptides indicated with the results as set out in TableI.

                                      TABLE I                                     __________________________________________________________________________    Frequency of Positive Antibody Responses                                      to Various HCG Peptide-Conjugates                                                                     Number of Rabbits                                                             Immunological                                                                          Neutralization of                            Peptide                                                                              Carrier    Immunized                                                                           Binding Responses                                                                      Biological Activity                          __________________________________________________________________________    35 Amino Acid                                                                        Bovine Gamma Globulin                                                                    10    10       6                                            111-145                                                                       Morgan et al.                                                                        Keyhole Limpet                                                                           10    5        *                                            Peptide II                                                                           Hemocyanin                                                             31 amino acid                                                                        Poly-D-L-Alanine                                                                         10    9        5                                            115-145                                                                       Morgan et al.                                                                        Bovine Serum Albumin                                                                     12    12       6                                            Peptide III                                                                   44 amino acid                                                                        Keyhole Limpet                                                                           10    8        *                                            105-148                                                                              Hemocyanin                                                             Peptide XV                                                                    Natural                                                                              Keyhole Limpet                                                                           10    10       *                                            109-145                                                                              Hemocyanin                                                             Keutman                                                                       Peptide XII                                                                   __________________________________________________________________________     *Additional time needed for assessment                                   

EXAMPLE XXIII

Antigen was prepared by reacting a diisocyanate (T.D.I.C.-see above)coupling reagent with carrier (tetanus toxoid), extracting excessreagent and incubating the activated carrier with the peptide ofStructure (II). Baboons were immunized with the antigen and results ofmating 4 animals three times are shown in FIG. 1. The data in FIG. 1show that from 12 exposures (matings) one pregnancy resulted even thoughrelatively low levels of immunity from the antigen were achieved.Non-immunized baboons of the same colony had a fertility rate ofapproximately 85%.

EXAMPLE XXIV

Referring to FIG. 2, baboons were immunized initially with a betasubunit of HCG modified by diazotization in a manner similar to thatdescribed in conjunction with Example II. Following this initialadministration, the baboons were injected 21 and 42 days later with thepeptide of Structure (II) above which had been modified by the samediazotization process. FIG. 2 shows plots representing the levels ofantibodies generated in consequence of these administrations. Suchquantities of antibodies are expressed as micrograms ofisotopically-labeled HCG that will bind each milliliter of serum fromthe baboons at specified days after the initial injection. The levelsshown were maintained for a period of over one year.

                  TABLE 2                                                         ______________________________________                                        Breeding of Immunized Baboons                                                  Diazo-β-HCG presensitized!                                              Booster: Diazo-β-HCG-(111-145)                                                  1             2                                                               Pre-Mate              Pre-Mate                                                Titer   Ovul.  Preg.  Titer   Ovul.                                                                              Preg.                               ______________________________________                                        Mating No. 1                                                                           5.00      +      -    4.20    +    -                                 Mating No. 2                                                                           4.25      +      -    4.10    +    -                                 Mating No. 3                                                                           4.22      +      -    4.00    +    -                                 Mating No. 4                                                                           4.17      +      -    3.89    +    -                                 Mating No. 5                                                                           3.80      +      -    3.76    +    -                                 Mating No. 6                                                                           6.65      +      -    5.00    +    -                                 Mating No. 7                                                                           5.90      +      -    4.75    +    -                                 Mating No. 8                                                                           5.10      +      -    4.20    +    -                                 Mating No. 9                                                                           5.00      +      -    4.25    +    -                                 Mating No. 10                                                                          4.66      +      -    4.00    +    -                                 ______________________________________                                    

In Table 2, the results of breeding the two baboons represented in FIG.2 are revealed in tabular form. The Table presents the results of matingthese animals ten times over a period of approximately one year. Thesedata suggest that the animals ovulated in every cycle; however, nopregnancy was observed, as indicated by the animal having a menstrualperiod at or before the expected time therefor. While the resultstabulated demonstrate the efficacy of the entire procedure, it wasobserved for the particular structure utilized in the primaryimmunization, i.e. Structure (II), antibody cross reactivity with LH wasobserved. Such cross reactivity may be avoided by the utilization of thefragment conjugation procedures set forth in detail hereinabove.

EXAMPLE XXV

The specificity of antibody response to a CG fragment-macromolecularcarrier is represented by the instant experiment. A 35 amino acidsequence Structure (II), herein "synthetic peptide"! of the HCG betasubunit was conjugated with ovine gamma-globulin and administered to ababoon. Varying doses of each of these three hormones were tested fortheir ability to compete with I¹²⁵ -labeled synthetic peptide ofStructure (II) bound to the anti-serum. The results are set forth inFIG. 3. Note from the data in this figure that human LH was ineffectivefor displacement of tracer antigen at doses up to 2.5 IU (internationalunits). Since HCG displaced antigen at a dose of 20 mIU, thecross-reactivity with HLH in this assay system was less than 0.8%.Baboon CG also displaced I¹²⁵ -labeled antigen in this assay and, basedon biological potency of the two hormones, was about 20% as effective asHCG.

EXAMPLE XXVI

The following experiments were carried out to determine whether thecarbohydrate chains contained in the C-terminal 37 residues of beta-MCGinfluence the immunogenicity of that peptide.

A peptide representing amino acid residues 109-145 of beta-HCG wasisolated from a chymotryptic digest of reduced and carboxymethylatedbeta-ECG by procedures reported by Keutmann, H. T.; Williams, R. M., J.Biol. Chem., 252, 5393-5397, (1977). This peptide is identified in Table3 below as P-1. The purity of the peptide was confirmed by amino acidand terminal end group analyses. A portion of the isolated peptide wastreated with anhydrous hydrofluoric acid (HF) to remove carbohydratemoieties and repurified by column chromatography according to methodsdescribed by Sakakibara S. et al., Bull Chem. Soc. Japan, 40, 2164-2167,(1967). This portion of the isolated peptide is identified in Table 3 asP-2. Complete removal of the sugar chains was confirmed by carbohydrateanalysis; see Nelson Norton, J. Biol. Chem., 153, 375-380, (1944). Athird peptide with the amino acid sequence 109-145 of beta-HCG wasprepared synthetically using the solid state synthesis procedure ofTregear, G. W. et al., Biochem., 16, 2817, (1977). This third peptide isidentified in Table 3 as P-3. Highly purified HCG was used in allimmunological experiments where reference was made to intact HCG.

PREPARATION OF IMMUNOGENS AND IMMUNIZATIONS

Conjugates of the three peptides to keyhole-limpet hemocyanin (KLH) wereprepared using tolylene diisocyanate. A peptide-carrier ratio of 4-6peptides per 100,000 daltons of carrier was obtained for differentconjugates prepared according to amino acid analyses. Rabbits wereimmunized with conjugates by three multiple site intramuscularinjections of 1.0 mg. of conjugate in 0.5 ml. of saline emulsified withan equal volume of Freund's complete adjuvant. Injections were given at3 week intervals and weekly blood samples were collected from 3-20 weeksof immunization.

EVALUATION OF ANTISERA

Antisera to all conjugates were monitored for antibody levels byreacting dilutions of sera with I¹²⁵ labeled HCG (Chloramine T method)at 4° C. for 5 days and precipitating immune complexes with sheepanti-rabbit gamma globulin serum. Antibody levels were determined byassessing dilution curves in which a linear correlation between dilutionand binding of labelled antigen at equilibrium occurred. At least 3points in each curve were used in calculating levels. These levels wereexpressed as micrograms of HCG bound per ml. of undiluted serumcalculated by multiplying mass of labelled antigen bound by serumdilution.

A radioimmunoassay system employing I¹²⁵ HCG and antisera raised topeptide conjugates was used to determine the relative ability of HCG andpeptides to compete with labeled HCG. Peak antibody levels from eachrabbit were evaluated in these studies. Antigens and antisera containedin phosphate-buffered saline (pH 7.4) BSA (1%) were added to test tubesand incubated at 4° for 5 days. Separation of free and bound tracer HCGwas accomplished by the addition of sheep anti-rabbit gamma globulinserum and then incubation for 48 hours followed by centrifugation.Assessment using methods described by Rodbard, D. in: Odell, W. D. andDaughaday, W. H., eds., "Competitive Protein Binding Assays", J. B.Lippincott, Phila, Pa. (1971). The ability of unlabeled HCG and peptidesto compete with I¹²⁵ MCG for antibody binding sites was expressed asmoles of unlabeled antigen, per mole of unlabeled HCG, required toreduce the binding of labeled HCG by 50%. For this purpose molecularweights for HCG, P-1, P-2, and P-3 of 38,000, 7,000, 3,990, and 3,990respectively were used. The molecular weight of the P-1 peptide was anestimate since the contribution of the carbohydrate chains to its sizewas not determined. Four radioimmunoassays were performed with each ofthe 11 antisera studied and the results presented as the mean of thefour values.

RESULTS

Parallel dose response curves of HCG and peptides were observed in allradioimmunoassays. In the assay system employed, 200-400 moles ofunlabeled HCG was required per mole of labeled HCG at 50% binding of thelatter to antisera. There was no detectable difference among antisera tothe 3 peptide conjugates in the ability of intact HCG to compete withlabeled hormone for antibody binding sites.

Data obtained from comparing the ability of HCG and peptides to competewith I¹²⁵ ECG for binding to anti-peptide sera revealed some qualitativedifferences in the antisera (Table 3). Much larger quantities of P-2peptide and P-3 peptide were required to reduce I¹²⁵ HCG binding thanwere required than of P-1 peptide when sera against the P-1 peptide weretested. While similar quantities of P-2 and P-3 peptides were requiredto inhibit one mole of labeled HCG binding, these were 2-10 times theamounts of the P-1 peptide required.

Differences in the quantities of peptides required to compete with anequivalent mass of labeled HCG were less using antisera raised tocarbohydrate-free natural peptide (P-2). More P-1 peptide was needed foran equal reduction in binding than of the other 2 peptides. Nosignificant difference could be detected in the quantities of P-2 or P-3peptides required among the 3 antisera tested.

Approximately 1.5-2.0 times as much P-1 peptide was required to competeequally with I¹²⁵ HCG for antibodies raised to the P-3 peptide but P-2peptide reacted nearly as well as did the synthetic peptide.

DISCUSSION

Despite low levels of antibodies obtained in this study, thecarbohydrate-containing peptide was not more immunogenic than thosewithout this moiety when conjugates to both were prepared in the samemanner.

From these studies, it can be concluded that although antibodies tocarbohydrate free peptides are qualitatively different from those to thenatural peptide, antisera generated to the synthetic peptide reactedwith HCG as well as anti-sera to natural peptides and equivalent tonatural and synthetic peptides elicited similar anti-HCG levels inrabbits.

                                      TABLE 3                                     __________________________________________________________________________    Mean Quantities of HCG and 109-145 C-Terminal β-HCG                      Peptides Required to Compete with I.sup.125 HCG at 50% Binding                of Labelled Hormone                                                           Unlabeled Antigens                                                            Antisera                                                                            HCG     P-1      P-2     P-3                                            Rabbit                                                                              mol/mol mol/mol  mol/mol mol/mol                                        __________________________________________________________________________    No.   HCG I.sup.125                                                                         HCG I.sup.125                                                                          HCG I.sup.125                                                                         HCG I.sup.125                                        (X ± SE)                                                                           (X ± SE)                                                                            (X ± SE)                                                                           (X ± SE)                                    Anti P-1                                                                      78    284                                                                              (12.6)                                                                             430 (11.8)                                                                             4565                                                                             (200.8)                                                                            3628                                                                              (154.1)                                    79    350                                                                              (13.5)                                                                             404 (18.5)                                                                             855                                                                              (33.4)                                                                             881 (42.2)                                     171   403                                                                              (17.7)                                                                             343 (9.9)                                                                              899                                                                              (35.1)                                                                             759 (37.1)                                     173   377                                                                              (16.5)                                                                             320 (13.9)                                                                             1448                                                                             (72.4)                                                                             1536                                                                              (73.7)                                     Anti P-2                                                                      93    247                                                                              (11.8)                                                                             385 (18.2)                                                                             264                                                                              (12.5)                                                                             268 (12.73)                                    94    294                                                                              (14.1)                                                                             431 (15.5)                                                                             362                                                                              (15.2)                                                                             329 (13.8)                                     252   201                                                                              (9.6)                                                                              296 (12.4)                                                                             216                                                                              (7.7)                                                                              205 (9.0)                                      Anti P-3                                                                      405   496                                                                              (23.6)                                                                             998 (47.4)                                                                             628                                                                              (27.6)                                                                             309 (13.6)                                     411   489                                                                              (20.5)                                                                             1200                                                                              (50.4)                                                                             678                                                                              (29.7)                                                                             413 (16.1)                                     416   364                                                                              (13.1)                                                                             581 (20.9)                                                                             400                                                                              (14.4)                                                                             271 (12.8)                                     417   340                                                                              (14.9)                                                                             474 (18.4)                                                                             176                                                                              (6.8)                                                                              105 (4.6)                                      __________________________________________________________________________

EXAMPLE XXVII

In this Example, a polypeptide fragment structure having an --SH groupis activated utilizing the following reagent of Formula B shown in FIG.9 in which X is a phenyl group substituted with a single methyl grouportho to the --CON₃ grouping. A solution of the reagent (1.2 eq. per--SH group in the polypeptide) in a suitable water miscible organicsolvent, such as dioxane, is added to a solution of the polypeptidefragment, e.g. Structure (XII) (which has had its amino groups blocked)in aqueous buffer at pH 6.5. After 2 hours, the solvent is removed at atemperature of less than 30° C. under vacuum, and to the residue areadded water and ethyl ether (1:1). The aqueous layer is separated andits pH adjusted to approximately 8.5 by the addition of sodium hydroxidesolution and this alkaline mixture is added rapidly to an aqueoussolution of the carrier, e.g. the above described influenza subunit,maintained at pH 8.5 by a suitable buffer. After a further 4 hours, theconjugate is isolated by gel filtration.

EXAMPLE XXVIII

With the following reagent of Formula C shown in FIG. 9 a solution orsuspension of a carrier containing no sulfhydryl groups (such asflagellin) in a suitable aqueous buffer at a pH 6.5 is treated with therequired (1.2 eq per --NH₂ desired to be reacted) amount of a solutionof the reagent in dimethylformamide. After 1 hour, the modified carrieris isolated by column chromatography and added to buffer at pH 6-7. Thisis then treated with a solution of the selected fragment (containingsulfhydryl groups) in the same buffer and the reaction is allowed toproceed for 12 hours before the conjugate is isolated by columnchromatography.

EXAMPLE XXIX

Modification of non-sulfhydryl containing peptide fragments e.g.Structure (II)! or a carrier such as flagellin to produce a sulfhydrylcontaining peptide via "thiolactonization" is carried out as follows:

The peptide is dissolved in a 1M aqueous solution of imidazolecontaining 0.5% of ethylenediaminetetraacetic acid at a pH of 9.3 underan atmosphere of nitrogen, and a 100 fold excess of N-acetylhomocysteinethiolactone is added in three portions at eight hour intervals. After atotal of 30 hours, the pH is adjusted to 3-4 with acetic acid and themodified peptide is isolated by gel chromatography and elution with 0.5Macetic acid.

EXAMPLE XXX

The carrier protein is reacted with the N-hydroxysuccinimide ester of ahalo-(either chloro, bromo or iodo) acetic acid in the general proceduredescribed in the first part of Example XXVII thus yielding a modifiedcarrier containing the required number of halomethyl alkylating groupsas desired.

To a solution of the sulfhydryl containing peptide e.g. Structure (XII)!in a phosphate buffer at pH 6.5-7.0 under nitrogen at room temperatureis added an aqueous solution or suspension of the modified carrierprepared above. The mixture is stirred for 12 hours. It is then washedwith ethyl acetate and the conjugate contained in the aqueous phase ispurified by dialysis, gel chromatography and lyophilization.

Should neither the carrier nor polypeptide fragment contain a sulfhydrylgroup, one may be introduced into either of them by the standardprocedures such as "thiolactonization" described above in Example XXIX.

EXAMPLE XXXI

This example illustrates the use of a modified polypeptide of theinvention in repressing fertility in baboons.

A polypeptide of Structure (XII) above, identical to the residues109-145 of beta-HCG, was prepared by total synthesis using the solidphase synthesis method described in Tregear et al, Biochem., 16, 2817,(1977). The purity of the peptide was assessed using thin-layerchromatography, high-voltage electrophoresis and amino acid analysis.The peptide was conjugated to the amino groups of tetanus toxoid via thecysteine residue at position 110 by the method described in Lee et al.,Mol. Immunol., 17, 749, (1980). The resultant conjugated polypeptidecontained 22 peptides per 100,000 daltons of the toxoid.

Male and female baboons (obtained from Primate Imports, Inc., PortWashington, N.Y.) were housed individually in metabolic cages measuring89×94×114 cm (L×W×H) for females and 168×94×165 cm for males, each cagebeing equipped with a "squeezebar" mechanism for restraining the baboon.Each male baboon was housed in a separate room in which were also housedsix to ten females. The room temperature was maintained at 21° C. withartificial light for 12 hours daily and the baboons were fed on PurinaMonkey Meal (product of Ralston Purina, St. Louis, Mo.) mixed withcorned beef, corn syrup and a vitamin mineral supplement; fresh fruitwas given daily as a conditioning aid and water was provided ad libitum.Daily observations were made of each female baboon to establish thepattern of sex skin turgescence/deturgescence and menstrual bleeding.

In order to ensure that the results of the test described below werestatistically valid, the number of baboons was determined using arcsinetransformation of the projected fertility rates and the resultant valuesapplied to probability tables, as described in Sokal et al., Biometry,W. H. Freeman and Co., San Francisco (1969), page 609. For an alphalevel of p=0.05 and a 90% confidence of detecting a significantreduction in fertility rate when the control rate is at least 70% andthe immunized is not greater than 10%, a group size of 15 animals pergroup was determined to be required and thus this was the group sizeused in the experiment.

Accordingly, 30 female baboons whose cycle length varied by no more thanthree days each side of its mean and who had exhibited progesteronelevels of at least 3.0 ng./ml. for each of their last three menstrualcycles (thus indicating ovulation) were randomly assigned to each of twogroups of 15 animals. Six male baboons, who had each proved theirfertility by siring several offspring were selected for use in theexperiments.

The control group of 15 baboons were immunized with pure tetanus toxoidwhile the other group received the aforementioned modified polypeptideconjugated with this toxoid. The antigens were dissolved inphysiological saline, mixed with an equal volume of Complete Freund'sAdjuvant (supplied by Difco Laboratories, Detroit, Mich.) and emulsifiedjust prior to each immunization. The modified polypeptide/toxoidconjugate was dissolved at a concentration of 4.0 mg/ml and a dose of2.0 mg given to each baboon in the second group, whereas the pure toxoidwas dissolved in a concentration 2.0 mg/ml and a dose of 1.0 mg. givento each baboon in the control group; since the conjugate comprisesapproximately 50% of the toxoid by weight, the dose of toxoidadministered to each animal was substantially the same. Each dose of thepure toxoid or of conjugate in the adjuvant, approximately 1.0 ml involume, was injected intramuscularly into four separate sites in theanimal, two in each thigh. The first (primary) immunization was givenduring the first five days of the menstrual cycle, with subsequentimmunizations given at 28 day intervals thereafter or until a pregnancywas confirmed. Females that did not become pregnant received five or siximmunizations during the course of the study depending upon the lengthof their individual menstrual cycles.

Blood samples were collected from the female baboons without anesthesiavia the cubital vein, five to six ml. of blood being drawn at weeklyintervals beginning at 21 days after the primary immunization, and alsoimmediately before and after mating for antibody determinations. Bloodsamples for progesterone determinations were drawn five and seven daysafter sex skin deturgescence and, in cycles in which mating occurred,samples for pregnancy testing were drawn daily commencing 12 days afterdeturgescence and continuing until pregnancy was confirmed ormenstruation began. After the blood samples were withdrawn, the serumwas removed and samples not immediately tested were stored at -20° C.

The serum samples thus obtained were tested for the presence ofantibodies to =¹²⁵ I-labelled HCG, Structure (XII), baboon chorionicgonadotropin (bCG) and tetanus toxoid by the methods described in Powellet al., Jr., Reprod. Immunol., 2, 1, (1980). As described in this Powellet al. paper, Structure (XII) can be labelled with ¹²⁵ I only afterintroduction of a tyrosine residue into the peptide. The HCGpreparation, which was highly purified, had a biological potency of10,800 IU/mg., while the bCG preparation, which was only partiallypurified, had a biological potency of 850 IU/mg. Concentrations oflabelled antigen capable of saturation of antibody combining sites atequilibrium were reacted with dilutions of serum for five days at 4° C.,followed by separation of the antigen-antibody complex from the unboundlabeled antigen using the double-antibody method. With the exception oftetanus toxoid, the concentrations of labeled antigens were adjusted sothat equimolar quantities were reacted with the serum. The molecularweight of bCG, which has not yet been established, was assumed to be thesame as that of HCG, namely 38,000. The labeled antigen binding for HCG,bCG and Structure (XII) was expressed as moles/liter (M/L)×10⁻¹⁰,whereas for tetanus toxoid, due to its molecular heterogeneity, bindingwas expressed as micrograms/ml.

All 30 female baboons were mated during the course of their thirdmenstrual cycle following the primary immunization and for the next twoconsecutive cycles if they did not become pregnant. Based upon theirpreviously established menstrual histories, each female baboon wastransferred to a male's cage three days prior to expected ovulation.Cohabitation was continued until the day of sex skin deturgescence, 2-3days after ovulation and the female was then transferred back to herindividual cage. If the first mating did not make the female pregnant,subsequent matings were conducted with a different male baboon.

The serum levels of steroid hormones and bCG were determined by theradioimmunoassay methods described in Powell et al., Clin. Chem., 19,210, (1973) and Hodgen et al., J. Clin. Endo. Metabol., 39,457, (1974).The assay for bCG was conducted with an antiserum to Ovine-LH-betasupplied by Dr. Gary Hodgen, Bethesda, Md.; the supplier has previouslyshown, by means of unpublished data, that the binding of ¹²⁵ I-HCG tothis antiserum can be displaced sensitively with bCG, but not withbaboon LH. The sensitivity of this assay was 5 mIU of HCG/ml. Sinceantibodies produced in the female baboons immunized with the Structure(XII) conjugate were capable of binding the labeled ECG used in the ECGassay, this assay system could not be used to determine pregnancy in thebaboons immunized with the conjugate. Accordingly, pregnancy testing ofthe conjugate-immunized baboons was performed by measuring estradiol andprogesterone levels only. In the tetanus toxoid-immunized baboons,however, pregnancy was tested using the CG assay as well as the steroidassays.

Data obtained on antibody levels, cycle levels and progesteroneconcentrations were evaluated by various methods for randomized designexperiments, as set out in Ostle, B., "Correlation Methods", inStatistics in Research, Ames I. A., Iowa State College Press (1954),page 174, while assessment of the mating data was accomplished by thechi-squared procedure set forth in Mantel, Cancer Chemotherapy Reports,50, 163, (1966); this procedure compares the mating data in its entiretyand not only in terms of individual matings.

RESULTS

The tetanus toxoid antibody level in both the baboons immunized with thepure tetanus toxoid and those immunized with the conjugate are shown inTable 4. High antibody levels against tetanus toxoid were produced inboth groups of baboons 60 days after primary immunization, with peaklevels being reached in 90-120 days. The differences between the tetanustoxoid antibody levels in the baboons immunized with the pure tetanustoxoid and with the conjugate were not significant at the p =0.05confidence level. Thus, it will be seen that, in addition to theantifertility effects observed below, the instant conjugate conferred asignificant degree of protection against tetanus. Accordingly, bycareful choice of the hapten to which the polypeptide is conjugated inthe instant modified polypeptide, the invention provides a method ofprotecting against a disease linked with the presence of the hapten aswell as against pregnancy.

                  TABLE 4                                                         ______________________________________                                        Levels of antibody produced by female baboons against Tetanus                 Toxoid from immunization with Tetanus Toxoid or Tetanus                       Toxoid conjugated with Structure (XII) synthetic peptide                      during the first five months of immunization.                                              Tetanus Toxoid Antibody Titer (micro-                            Baboons      grams/ml) Days after Primary                                     Immunized    Immunization*                                                    With         30      60      90    120   150                                  ______________________________________                                        Tetanus Toxoid                                                                         x =     20.6    515.7 662.9 667.2 821.6                                       se =    7.3     37.8  46.5  73.3  --                                          n =     15      15    15    5     1                                  Conjugate                                                                              x =     21.8    404.8 608.9 649.7 621.3                                       se =    10.4    39.8  29.2  28.7  30.9                                        n =     15      15    15    14    13                                 ______________________________________                                         *Actual day may vary ± 5 days                                         

                                      TABLE 5                                     __________________________________________________________________________    Antibody levels produced by female baboons against Structure (XII),           baboon chorionic                                                              gonadotropin and human chorionic gonadotropin from immunization with          conjugate.                                                                    Values were determined from serum collected during the early luteal phase     of each                                                                       menstrual cycle.                                                              Antibody     Antibody Level (M/L × 10.sup.-10)                          Reactive     Menstrual Cycle                                                  To           1    2     3     4     5                                         __________________________________________________________________________    Structure (XII)                                                                       x =  62.8 246.3 195.1 184.8 204.5                                             95% CI =                                                                           28.2-97.6                                                                          29.9-462.3                                                                          82.0-308.2                                                                          57.1-312.6                                                                          37.3-371.7                                Baboon Chorionic                                                                      x =   2.6  7.3   10.4  10.0  9.9                                      Gonadotropin                                                                          95% CI =                                                                           1.1-4.0                                                                            3.1-11.5                                                                            3.1-17.8                                                                            1.8-16.7                                                                            0.02-19.5                                 Human Chorionic                                                                       x =  43.8 185.7 145.8 125.1 135.5                                     Gonadotropin                                                                          95% CI =                                                                           20.9-66.6                                                                          27.2-344.1                                                                          48.7-243.1                                                                          25.3-224.9                                                                          15.2-255.7                                        n =  15   15    15    14    13                                        __________________________________________________________________________

The mean antibody levels produced to HCG, bCG and Structure (XII) in thegroup of baboons immunized with the conjugate are shown in Table 5.Antibody levels to Structure (XII) reached a maximum during the lutealphase of the second menstrual cycle, approximately 60 days after theprimary immunizations, as did antibodies to HCG. While the mean antibodylevel to HCG and Structure (XII) were maintained by repeated boosterimmunizations, the responses of individual baboons varied considerably.There was a very close correlation between antibody levels to HCG andStructure (XII), r=0.97. The mean levels of antibodies which reactedwith HCG were only 71% of those reacting with Structure (XII). However,because of variation in levels between the individual animals, thisdifference in levels is not significant at the p=0.05 level. Althoughthe means levels of antibody reacting with bCG were only 4.5% of thosereacting with Structure (XII) and 6.3% of those reacting with HCG, thesebCG antibody levels reached maximum levels by the first mating cycle andremained close to that level during the next two cycles. There is asignificant positive correlation (r=0.78) between the bCG antibody leveland the Structure (XII) antibody level during these three cycles.

                  TABLE 6                                                         ______________________________________                                        Levels of antibody produced by female baboons against hCG and                 baboon CG from Immunization with conjugate. Levels determined                 from serum obtained during the luteal phase of three                          consecutive mating cycles.                                                    Antibody Titer - M/L × 10.sup.-10                                       Mating Cycle 1  Mating Cycle 2                                                                             Mating Cycle 3                                   Baboon hCG      bCG     hCG    bCG   hCG    bCG                               ______________________________________                                        1      42.3     6.2     31.5   7.4   29.4   3.2                               2      35.0     2.9     40.0   3.6   40.1   3.0                               3      117.3    11.6    151.2  11.9  188.8  12.7                              4      193.2    21.2    108.9  11.6  25.2   3.0                               5      469.8    52.6    598.2  51.4  602.4  60.7                              6      479.8*   0.9*                                                          7      9.8      1.1     10.3   0.9   12.2   1.2                               8      10.8     1.7     11.2   1.4   9.7    1.0                               9      72.4     4.4     68.2*  1.7*                                           10     470.9    15.5    427.4  14.6  536.3  16.0                              11     64.1     17.6    49.5   9.5   25.2   4.6                               12     50.0     5.1     21.6   2.6   63.4*  1.4*                              13     46.6     4.3     57.6   6.3   59.2   6.4                               14     41.0     2.7     127.6  13.2  110.2  12.0                              15     85.2     8.4     48.5   4.1   60.3*  1.6*                              ______________________________________                                         *Pregnancy resulted from mating                                          

Moreover, the antibody levels for HCG and bCG shown in Table 6 revealsignificant correlation between these two antibody levels during thethree mating cycles. The correlation coefficients (r) are 0.55, 0.89,and 0.85 for the first, second and third mating cycles respectively, thelatter two correlation coefficients being significant at the 1% level.

Table 7 below compares the cycle lengths and progesterone levels in theluteal phase of the menstrual cycles before and after the immunizationsof the two groups of baboons. The pre-immunization portion of this tableshows that the two randomly assigned groups of baboons showed nosignificant differences at the p=0.05 level in the cycle length orprogesterone levels for the three cycles immediately proceedingimmunization. Even though both groups of baboons were immunized usingComplete Freund's Adjuvant, no change significant at the p=0.05 level inthe cycle lengths or progesterone levels was apparent when the threepre-immunization cycles were compared with the post-immunization cycles.

                                      TABLE 7                                     __________________________________________________________________________    Comparison of the length of menstrual cycles and luteal phase                 progesterone                                                                  levels before and during the course of immunization of female baboons         with                                                                          Tetanus Toxoid or conjugate.                                                  Baboons    Menstrual Cycles                                                   Immunized  Pre-Immunization                                                                        Post-Immunization                                        With       -3 -2  -1 1   2  3   4  5                                          __________________________________________________________________________    Tetanus Toxoid                                                                Cycle length                                                                         x   33.1                                                                             32.2                                                                              32.9                                                                             33.0                                                                              32.0                                                                             32.0                                                                              29.0                                                                             --                                                se  0.8                                                                              0.8 0.8                                                                              1.5 1.0                                                                              1.1 -- --                                                n*  15 15  15 15  15 5   1  --                                         Progesterone                                                                         x** 6.3                                                                              7.1 7.2                                                                              7.0 7.9                                                                              8.6 10.3                                                                             6.7                                               se  0.6                                                                              0.6 0.6                                                                              0.9 1.0                                                                              0.6 0.8                                                                              --                                                n   15 15  15 15  15 15  5  1                                          Conjugate                                                                     Cycle length                                                                         x   32.0                                                                             32.4                                                                              32.5                                                                             32.1                                                                              33.3                                                                             33.5                                                                              34.6                                                                             32.5                                              se  0.6                                                                              0.7 0.7                                                                              0.9 1.1                                                                              1.1 1.3                                                                              0.6                                               n*  15 15  15 15  15 14  13 11                                         Progesterone                                                                         x** 7.3                                                                              7.9 7.6                                                                              7.1 7.3                                                                              7.6 7.7                                                                              7.3                                               se  0.7                                                                              0.7 0.6                                                                              1.0 0.9                                                                              0.8 0.6                                                                              0.3                                               n   15 15  15 15  15 15  14 13                                         __________________________________________________________________________     *decreases as a result of pregnancy                                           **includes values for cycle in which pregnancy occurred                  

                  TABLE 8                                                         ______________________________________                                        Comparison of fertility rates for female baboons immunized with Tetanus       Toxoid to those immunized with conjugate. Matings commenced during            the course of the third menstrual cycle following primary immunization.       Baboons                Total    Total Overall                                 Immunized Mating Cycle Number   Number                                                                              Fertility                               With      1      2      3    Pregnant                                                                             Mated Rate (%)                            ______________________________________                                        Tetanus Toxoid                                                                Number Mated                                                                             15*   5      1           21                                        Number Pregnant                                                                         10     4      1    15                                               Fertility Rate (%)                                                                      66.7   80.0   100.0             71.4                                Conjugate                                                                     Number Mated                                                                             15*   14     13          42                                        Number Pregnant                                                                         1      1      2    4                                                Fertility Rate (%)                                                                       6.7   7.1    15.4              9.5                                 ______________________________________                                         *Includes 1 anovulatory menstrual cycle                                  

Table 8 shows the highly significant difference in the fertility ratebetween the two groups of baboons. On the first mating, 10 out of 15 ofthe tetanus toxoid-immunized baboons became pregnant, 4 of the 5remaining baboons became pregnant after the second mating and the singleremaining baboon became pregnant after the third mating. Thus, of 21matings, 15 resulted in pregnancy, giving a fertility rate of 71.4%.

Of the 15 baboons immunized with the conjugate, one became pregnantafter the first mating, one of the remaining 14 became pregnant afterthe second mating and 2 of the remaining 13 baboons became pregnantafter the third mating. Thus, 42 matings resulted in four pregnanciesfor a fertility rate of 9.5%.

Chi-squared analysis of this data shows that this difference infertility rate is highly significant (p less than 0.005) even afteradjustment for the small sample size.

The antibody levels to tetanus toxoid from sera obtained during thethree mating cycles were assessed for correlation to the outcome ofmating for all animals. No correlation significant at the p=0.05 levelwas found for either group of baboons. Similarly, although thepost-mating antibody levels for menstrual cycles 3, 4, and 5 were quitevariable, as shown by the rather large 95% confidence interval, nocorrelation significant at the p=0.05 level was found between theantibody levels to Structure (XII), HCG or bCG and the fertility of theconjugate-immunized group. However, there was a significant difference(p less than 0.025) between the mean bCG antibody level in the fourpregnant conjugate immunized baboons (1.4×10⁻¹⁰ M/L) and the mean (9.8)and the 95% confidence interval (5.5-14.1) levels of the same antibodiesfor all matings, thus suggesting that these four baboons became pregnantbecause their bCG antibody levels were insufficiently raised.

I have shown in the following published papers: Excerpta MedicaInternational Congress Series No. 402, pp. 1379 (1976); and"Physiological Effects of Immunity Against Reproductive Hormones", R. G.Edwards and M. H. Johnson (ed.), Cambridge University Press, p. 249(1975); that immunizations with synthetic peptides containing aC-terminal portion of beta-HCG result in the production of antibodiescapable of binding and neutralizing the biological activity of intactHCG and that these antibodies exhibit a low degree of reactivity withbaboon CG. This is in accord with the results of this Example, in whichthe conjugate based on Structure (XII), the 109-145 sequence ofbeta-HCG, produced high levels of antibody to HCG but relatively lowlevels of antibody to bCG. Nevertheless, despite the low levels of bCGantibody, the conjugate was highly effective in preventing pregnancy.The pregnancy-preventing action of the conjugate demonstrated in thisExample provides the statistically valid proof of the feasibility ofthis approach to fertility regulation in humans. It may reasonably beanticipated that the antifertility effects which the conjugate wouldproduce in humans would be considerably greater than that in baboons,given the much higher level of antibodies to HCG produced in thebaboons, as compared to levels of antibody to bCG.

The exact mechanism of action of the conjugate is not known, althoughpresumably antibody neutralization of CG occurs in the peripheral bloodsoon after implantation and disrupts trophic hormone support to thecorpus luteum of pregnancy and causes early abortion. However, since theduration of the menstrual cycle is not significantly effected, itappears that pregnancy must be disrupted almost immediately afterimplantation.

EXAMPLE XXXII

The following experiments were conducted to determine the mostappropriate peptide and carrier for use in a modified polypeptide of theinvention intended for provoking antibodies to HCG.

The following peptides, each having a sequence derived at least in partfrom that of β-HCG were prepared by the same method as in Example XXXI(the numbers given refer to the sequence of bases in the full β-subunitof HCG, Structure (I) above:

a. 138-145, hereinafter referred to as Structure (XVI)Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;

b. 126-145, hereinafter referred to as Structure (XVII)Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;

c. 115-145, Structure (VI) above;

d. 111-145, Structure (II) above;

e. 109-145, Structure (XII) above;

f. 106-145, hereinafter Structure (XVIII)His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;

g. 105-145, hereinafter Structure (XIX)Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;

h. Cys-(Pro)₆ -(111-145), Structure (XIV) above;

i. (111-145)-(Pro)6-Cys, Structure (X) above; and

j. Cys-(105-145), with the SH group of the Cys residue at position 110blocked with an acetamidomethyl (ACM) group, hereinafter referred to asStructure (XX).

The peptides of Structures (XVIII), (XIX) and (XX) contained a protectedsulfhydryl group at the 110 cysteine position. The purity of all thepeptides was demonstrated using high-voltage electrophoresis, thin-layerchromatography and amino acid analysis.

The following carriers were used in the experiments: tetanus toxoid,polymerized flagellin, poly-DL-alanine/lysine (polyalanine), (poly DAL)poly(tyrosine, glutamic acid)/poly(alanine, lysine) (TGAL) andpolymerized sucrose (Ficoll), described above. The carriers used werediphtheria toxoid obtained from Connaught Laboratories, Swiftwater, Pa.)bovine gamma globulin (obtained from Swartz-Mann Laboratories,Orangeburg, N.Y.), Corynebacteriumparvum (obtained fromBurroughs-Wellcome, London, England), uncapsulated meningococcal proteinand pneumococcus polysaccharide.

A thiol group on each peptide was coupled to an amino group on thecarrier by the same method as in Example XXXI. The site at which thepeptide was coupled to the carrier depended upon the point at which athiol group existed or could be created on the peptide. Structure (XII)contains a cysteine at position 110 and accordingly, this peptide wasconjugated by means of the thiol group produced at this position aftercleavage of the synthetically produced disulfide dimer of the peptide.Structures (XVI), (XVII), (XVIII) and (XIX) had a thiol group introducedat the amino terminal group and were coupled to the carrier via thisintroduced thiol group. Structures (II) and (VI) had thiol groups at theterminal amino group and also at the amino group at the lysine residueat position 122; under the conditions used, approximately equal numbersof peptides were attached to the carrier at each of the two attachmentsites. Structures (X) and (XIV) were coupled to carriers using the thiolgroup on the terminal cysteine residue.

After the coupling of the peptide to the carrier, the resultingconjugates were all purified by gel filtration or ultrafiltration andthe ratio of the peptide to carrier was determined.

The animals used in these experiments were genetically beterogenousrabbits of the New Zealand White variety, weighing 2-4 kg. and inbredfemale mice of the C3H/He strain, 8-10 weeks old or retired breeders,weighing 20-30 gm. each and obtained from Jackson Laboratories, BarHarbor, Me. For use in the rabbits, the conjugates were dissolved insaline and emulsified with an equal volume of Complete Freunds'adjuvant, exactly as in Example XXXI. However, for immunization intomice, an adjuvant was prepared by mixing 1.5 parts by volume of ArlacelA (obtained from Hilltop Research, Miamiville, Ohio) with 8.5 parts byvolume of Klearol (Bate Chemical, Don Mills, Ontario, Canada) andautoclaving for 20 minutes at 15 lb. pressure. Thereafter, heat-killeddesiccated acetone-washed BCG bacteria (obtained from ConnaughtLaboratories, Willowdale, Ontario, Canada) were added at the rate of 5mg. per 10 ml. of adjuvant. Apart from the change of adjuvant, thesolution used to immunize the mice was prepared in the same way as thatused to immunize the rabbits.

The mice were immunized three times, the primary immunization beinggiven at day 0 with booster immunizations given at 21 and 38 days; thedose of conjugate injected on each occasion into mice was usually 100μg, though multiples of this dose were given where stated. Blood sampleswere collected from the mice at weekly intervals starting at day 14.

The rabbits were immunized three times at 21 day intervals (the primaryimmunization at day 0 and booster immunizations at 21 and 42 days), 1mg. of the conjugate being given at each immunization. Blood sampleswere collected from the rabbits weekly beginning at day 21. The sera ofthe blood samples drawn from both the mice and the rabbits wereseparated from the cells and stored at -20° C. prior to analysis.

The levels of antibody to both the peptide used and HCG were measured byisotopically labeling the antigen with ¹²⁵ I and reacting it withvarious dilutions of the antisera. In most cases, 250 pg. of labeledantigen was incubated with 200 μl of diluted serum for 120 hours at 4°C. Antigen binding was determined at three or more serum dilutions usinga double antibody technique, the results being expressed as nanograms(ng) of antigen bound per milliliter of undiluted serum. The minimumsensitivity was 1 ng/ml. In some experiments, the level of antigenbinding was tested using two concentrations of antigen with twodilutions of serum and expressing the results as M/L×10⁻¹⁰ by the samemethods as in Example XXXI.

The antigen binding levels found in the various groups of rabbits andmice were compared using the two-tailed Mann-Whitney U-test described byS. Siegel, "The Case of Two Independent Variables", in NonparametricStatistics, McGraw-Hill Book Company, New York (1956), p. 116. Resultswhere p<0.05 were considered significant. In mice, in a few cases wherethe level of antigen binding was too low to be detected by the methodsused, an arbitrary value of 0.1 ng/ml was assigned for statisticalcomparisons of values that were undetectable.

RESULTS

Mice were injected by the procedures set out above with doses of 100,40, 8 and 1.6 μg. of conjugates of Structure (II) and (XII) with tetanustoxoid. Table 9 below shows the levels of antibodies to both the peptideused and HCG at 21 and 35 days after the primary immunization. Analysisof these results shows that the differences between peptide binding inthe sera between the mice injected with the two conjugates weresignificant at 21 days but not at 35 days. An increase in antibodylevels to peptides was observed at 35 days with increasing doses ofantigen injected, but again there was no significant difference betweenthe antibody levels of the mice injected with the two conjugates at thesame dosage levels.

                                      TABLE 9                                     __________________________________________________________________________              Sera 21 days after  Sera 35 days after                              Tetanus Toxoid                                                                          Primary Immunization                                                                              Primary Immunization                            Coupled To Peptide                                                                      Immunization Dose (mg)                                                                            Immunization Dose (mg)                          of Structure                                                                            100  40   8    1.6  100   40   8    1.6                             __________________________________________________________________________              Antibody Level -- Peptide ng/ml (sd)                                II        10.4 12.6 8.6  4.2  38.0  27.4 16.4 5.8                                       (3.4)                                                                              (8.1)                                                                              (3.0)                                                                              (2.6)                                                                              (60.7)                                                                              (15.2)                                                                             (21.2)                                                                             (4.1)                           XII       0.1  0.9  1.1  0.1  207.8 80.3 85.0 9.0                                       (0)  (1.7)                                                                              (2.2)                                                                              (0)  (210.8)                                                                             (56.3)                                                                             (99.2)                                                                             (11.5)                          p, U-test 0.008                                                                              <0.016                                                                             0.008                                                                              0.008                                                                              0.112 0.190                                                                              0.548                                                                              0.392                                     Antibody Level -- HCG ng/ml (sd)                                    II        0.1  0.1  0.1  0.1  0.8   3.8  0.1  0.1                                       (0)  (0)  (0)  (0)  (1.4) (3.8)                                                                              (0)  (0)                             XII       0.1  0.1  0.1  0.1  51.4  2.3  9.6  1.1                                       (0)  (0)  (0)  (0)  (88.6)                                                                              (2.0)                                                                              (14.4)                                                                             (2.2)                           p, U-test >1   >1   >1   >1   0.286 0.730                                                                              <0.056                                                                             0.690                           __________________________________________________________________________

To determine the effect of chain-length of the peptide on antibodyresponse, groups of four rabbits were injected with conjugates oftetanus toxoid with the peptides of Structures (XVI), (XVII), (VI),(II), (XII), (XVIII), and (XIX). Sera from animals immunized with theseconjugates were reacted separately with equimolar quantities of labeledpeptide and labeled HCG, and the proportion of antibodies reactive topeptides which were also reactive to HCG determined. The results areshown in FIG. 4.

Contrary to what might be expected, the maximum reactivity to HCG is nota simple function of the chain-length of the peptide. Maximum reactivityto peptide and HCG was obtained by rabbits receiving the conjugate ofthe peptide of Structure (XII), representing residues 109-145 of β-HCG.The peptide of Structure (II), representing residues 111-145 of β-HCG,produced antibodies nearly as reactive as the peptide of Structure(XII), but the antibody levels produced by the longer peptides ofStructures (XVIII) and (XIX), representing respectively residues 106-145and 105-145 of β-HCG, were lower than those produced by the peptides ofStructures (XII) and (II). Not surprisingly, the shorter peptides alsoresulted in a lower proportion of antibody reacting to HCG.

A further series of tests were effected to determine the effect of thehexaproline cystine spacer sequences in the peptides of Structures (XIV)and (X) on antibody production. Conjugates of tetanus toxoid wereprepared coupled to the peptides of Structures (II) (the 111-145sequence without any spacer), (XIV) (the 111-145 sequence with aN-terminal spacer) and (X) (the 111-145 sequence with a C-terminalspacer), all conjugates containing a peptide:carrier ratio ofapproximately 20-22 peptides/10⁵ daltons of toxoid.

Table 10 below shows the antibody levels to HCG and peptides obtained inmice. Twenty-one days after primary immunization, the HCG antibodieswere significantly higher in the mice immunized with the conjugatescontaining either of the spacer peptides than with the non-spacerpeptide, Structure (II). Thirty-five days after primary immunization,the HCG antibody levels in mice immunized with the conjugate containingthe C-terminal spacer peptide of Structure (X) were significantlygreater than those of mice immunized with the non-spacer peptide, butthe difference in antibody levels between the latter and the micereceiving the conjugate of the N-terminal spacer peptide of Structure(XIV) was not significant.

                                      TABLE 10                                    __________________________________________________________________________              Sera 21 days after Primary Immunization                                                               Sera 35 days after Primary                                                    Immunization                                Tetanus Toxoid                                                                          Antibody    Antibody    Antibody    Antibody                        Coupled to Peptide                                                                      Level to                                                                            U-test                                                                              Level to                                                                            U-test                                                                              Level to                                                                            U-test                                                                              Level to                                                                            U-test                    of Structure                                                                            Peptide*                                                                            p     HCG*  p     Peptide*                                                                            p     HCG*  p                         __________________________________________________________________________    (a) II    10.4        0.1         38.0        0.8                                       (3.4)       (0)         (60.7)      (1.4)                           U-test (a):(c)  0.31-0.42   0.008       0.73-0.90   0.032                     (b) XIV   0.1         4.5         1.7         33.2                                      (0)         (5.2)       (1.8)       (48.0)                          U-test (b):(a)  0.170       0.008       0.032       0.286                     (c) X     1.2         3.7         0.4         1.2                                       (1.6)       (5.0)       (0.8)       (1.6)                           U-test (c):(b)  0.69-0.84   0.31-0.42   0.056       0.310                     __________________________________________________________________________     *nanograms/ml (sd)                                                       

However, very different results were obtained when the serum antibodieswere evaluated in terms of their ability to bind peptides. More of thelabelled peptide was bound by the antibodies produced by the conjugateof the non-spacer peptide of Structure (II) 35 days after the primaryimmunization than by the antibodies produced by mice immunized with theconjugate of the N-terminal spacer peptide of Structure (XIV). There wasno significant difference between the peptide binding abilities of thesera from mice immunized with the conjugates of the N-terminal andC-terminal peptides of Structures (XIV) and (X) respectively at thistime.

The tests in rabbits were carried out with the same three conjugates asin mice, and also with the conjugate of tetanus toxoid and the peptideof Structure (XII) (the 109-145 sequence of β-HCG) used in the precedingseries of tests. The antibody levels 10-13 weeks after the primaryimmunization are shown in FIG. 5. These results show that the meanantibody levels in rabbits immunized with the conjugate non-spacerpeptide of Structure (II) were lower than those of rabbits immunizedwith the spacer peptides of Structures (XIV) and (X), but the rabbitsinjected with the conjugate of the peptide of Structure (XII) had meanantibody levels to HCG comparable to the rabbits immunized with theconjugates of the spacer peptides of Structures (XIV) and (X).

A further series of tests was carried out to determine the effects ofdifferent carriers on antibody production. The peptide of Structure(XII), representing the 109-145 sequence of β-HCG, was coupled tovarious carriers in a ratio of 15-28 peptides per 10⁵ daltons of carrierand mice and rabbits were immunized with these conjugates. The antibodylevels to the peptide and to HCG were tested in the sera 21 and 35 daysafter the primary immunization. The results obtained in mice are shownin Table 11 below. Although the large standard deviations makes thedetection of significant differences difficult, the results do show thatthe tetanus toxoid conjugate elicited antibody levels to both thepeptides and HCG which were significantly higher than those produced bythe conjugates of all the other carriers. Mean antibody levels in thegroups injected with the peptide conjugated with flagellin and bovinegamma globulin were higher than those in which the peptide was linked tosynthetic sugar (Ficoll) or polypeptide carriers. Table 12 shows theU-test analysis of the data presented in Table 11.

                  TABLE 11                                                        ______________________________________                                        Sera 21 days after Sera 35 days after                                         Primary Immunization                                                                             Primary Immunization                                       Antibody Level to  Antibody Level to                                          Carrier                                                                              Peptide   HCG       Peptide  HCG                                       Used   ng/ml  (sd)   ng/ml                                                                              (sd) ng/ml                                                                              (sd)  ng/ml                                                                              (sd)                           ______________________________________                                        Tetanus                                                                              11.3   (5.1)  1.9  (2.1)                                                                              224.1                                                                              (139.0)                                                                             89.2 (128.2)                        Toxoid                                                                        Flagellin                                                                            5.5    (4.3)  0.7  (1.4)                                                                              39.8 (19.9)                                                                              24.7 (11.8)                         TGAL   2.9    (6.2)  2.7  (1.4)                                                                              15.0 (10.0)                                                                              10.9 (6.5)                          bov GG 3.5    (3.2)  0.1  (0)  43.3 (22.1)                                                                              26.7 (52.1)                         Poly-  0.5    (1.0)  0.1  (0)  3.1  (6.2) 0.5  (0.9)                          alanine                                                                       Ficoll 0.1    (0)    0.8  (1.6)                                                                              9.2  (5.7) 23.6 (43.3)                         None   1.3    (1.2)  0.1  (0)  0.8  (0.7) 0.1  (0)                            ______________________________________                                    

                                      TABLE 12                                    __________________________________________________________________________                Sera 21 days after Primary Immunization                                                               Sera 35 days after Primary                                                    Immunization                              Carrier     TT    Flagellin                                                                           TGAL  bovGG TT  Flagellin                                                                           TGAL bovGG                      __________________________________________________________________________    Antibody Level-Peptide                                                        Tetanus Toxoid                                                                            --    0.096 0.056 0.032 --  0.036 0.036                                                                              0.036                      Flagellin   0.096 --    0.310 0.42-0.55                                                                           0.036                                                                             --    0.016                                                                              1.000                      TGAL        0.056 0.310 --    0.310 0.036                                                                             0.016 --   0.056                      bov GG      0.032 0.42-0.55                                                                           0.310 --    0.036                                                                             1.000 0.056                                                                              --                         Antibody Level-HCG                                                            Tetanus Toxoid                                                                            --    0.310 0.31-0.42                                                                           0.150 --  1.000 0.250                                                                              0.250                      Flagellin   0.310 --    0.056 0.690 1.000                                                                             --    0.056                                                                              0.222                      TGAL        0.31-0.42                                                                           0.056 --    0.008 0.250                                                                             0.056 --   0.548                      bov GG      0.150 0.690 0.008 --    0.250                                                                             0.222 0.548                                                                              --                         __________________________________________________________________________

The results obtained in rabbits are shown in Table 13 below. The highestantibody levels were obtained in the rabbits immunized with conjugatesof bovine gamma globulin, tetanus toxoid and diphtheria toxoid, therebeing no significant difference between the peak antigen titers of thesethree carriers. Significantly lower antibody levels were found inrabbits immunized with bacterial carriers, while synthetic polypeptideand sugar carriers produced antibody levels which were significantlylower than those of bacterial carriers.

                  TABLE 13                                                        ______________________________________                                        Maximum Antigen Binding Levels*                                                            HCG        Peptide                                               Carrier Used   M/L × 10.sup.-10                                                                  sd     M/L × 10.sup.-10                                                                sd                                    ______________________________________                                        bov GG         479.2.sup.a                                                                             402.5  552.3.sup.a                                                                           410.8                                 Diphtheria Toxoid                                                                            365.3.sup.a                                                                             176.8  423.3.sup.a                                                                           189.3                                 Tetanus Toxoid 339.3.sup.a                                                                             95.5   417.6.sup.a                                                                           106.2                                 C-parvum       215.9     121.5  255.4   131.6                                 Flagellin      210.4     157.5  244.6   198.1                                 Pneumococcus Polysaccharide                                                                  135.7     102.8  153.7   113.0                                 Meningococcal Protein                                                                        97.9      107.6  118.4   122.6                                 TGAL           29.9      22.6   37.3    35.5                                  Ficoll         10.6      8.3    13.0    9.0                                   Polyalanine    8.6       4.7    12.2    7.5                                   ______________________________________                                         *4 rabbits/groups                                                             .sup.a not significantly different (p > 0.05)                            

A final series of experiments were performed to determine the effect ofthe peptide:carrier ratio on antibody production. Conjugates of tetanustoxoid and the peptide of Structure (XII) with peptide:carrier ratios offrom 5-33 peptides per 10⁵ daltons of carrier were prepared and groupsof four rabbits were immunized with these conjugates. The mean antibodylevels to HCG produced 42, 63, and 84 days after primary immunizationare shown in FIG. 6. Statistical analysis of the data in FIG. 6 shows nosignificant difference with peptide:carrier ratio 42 days after primaryimmunization, but 63 days after primary immunization the antibodyresponses to conjugates containing 23 or more peptides per 10⁵ daltonsof toxoid are significantly greater than those of conjugates with lowerpeptide:carrier ratios. A similar comparison 84 days after primaryimmunization shows that the antibody levels produced by conjugatescontaining 16 or more peptides per 10⁵ daltons of carrier aresignificantly greater than those of conjugates with a lowerpeptide:carrier ratio. Accordingly, it is believed that it isadvantageous to use a conjugate containing between 20 and 30 peptidesper 10⁵ daltons of carrier.

When mice were immunized with the same conjugates, the responses weremore variable and no linear dose-antibody response was observable, butthe highest antibody levels were obtained in mice receiving conjugatescontaining 28-23 peptides per 10⁵ daltons of carrier.

The above results show that antibodies formed to peptides with 30 ormore amino acids bind HCG better than those to peptides with fewerresidues. However, since the 40 and 41 residue peptides (Structures(XVIII) and (XIX) above) were not as reactive to HCG as the 35 or 37residue peptides of Structures (II) and (XII) above, it appears that noimmunological determinant of HCG is present in the 105-109 region of thebeta subunit thereof. Based upon the foregoing results, the preferredpeptides for use in forming conjugates to produce antibodies to HCG arethe peptides of Structures (XII) (the 109-145 sequence without spacers),(XIV) and (X) (the 111-145 sequence with N-terminus and C-terminusspacers respectively). The addition of the seven-residue spacer sequenceto either the N-terminus or the C-terminus of the 111-145 peptide ofStructure (II) produced higher antibody levels than the same peptidewithout spacer. It appears likely that a similar advantage can beproduced by attaching similar spacer sequences to the 109-145 peptide ofStructure (XII) since this peptide without spacers elicited responsessimilar to the 111-145 peptide with spacer (cf. results for Structures(XIV) and (XII)). On the other hand, it appears disadvantageous toattach peptides to the carrier at both position 122 and the N-terminus(see results from peptides of Structures (II) and (VI) above) sincepeptides attached to the carrier at both positions did not elicit levelsequivalent to those attached at either terminus alone. Probably couplingof the peptide at both its midpoint and its N-terminus affects itsconformation and creates an immunological determinant dissimilar to thatfound on intact HCG.

Moreover the results presented above strongly suggest that the bestcarriers for use in humans or other primates are tetanus toxoid anddiphtheria toxoid. While the antibody levels in rabbits for bovine gammaglobulin, tetanus toxoid and diphtheria toxoid are not significantlydifferent, the antibody levels produces in mice with conjugates of thebovine gamma globulin are not as high as those produced by conjugates ofthe two toxoids. Immunization of humans or other primates with tetanusand diphtheria toxoids is acceptable and even advantageous (since asingle vaccination can then provide protection against tetanus ordiphtheria as well as an isoimmunogenic action), whereas injections ofnon-primate gamma globulins may not prove safe. Conjugates of eithertetanus toxoid or diphtheria toxoid with a peptide:carrier ratio of20-30 peptides per 10⁵ daltons of carrier evoked large titers ofantibody reactive to HCG and would therefore appear to be suitable foran anti-HCG vaccine.

EXAMPLE XXXIII

This example illustrates the variations in antibody levels produced bychanges in the adjuvant and vehicle used in conjunction with a modifiedpolypeptide of the invention.

Based upon the results in Example XXXII above, the conjugates of tetanustoxoid with the peptides of Structures (XIV) and (XII) were selected asmost efficacious in generating antibodies to HCG and were thus used inthese experiments to select the optimum adjuvant and vehicle. Thetetanus toxoid/peptide conjugates were prepared in exactly the samemanner as in Example XXXII and purified by gel filtration andlyophilization. The conjugate of the peptide of Structure (XII)contained 21-25 peptides per 10⁵ daltons of carrier, while the conjugateof the peptide of Structure (XIV) contained 20-27 peptides per 10⁵daltons of carrier. A further conjugate was prepared by conjugating thesame tetanus toxoid to both the peptide of Structure (XIV) and thesynthetic muramyl dipeptide CGP 11637 (manufactured by Ciba-GeigyLimited Basle, Switzerland, -Formula (a) below). Using carbodiimide, thecarboxyl group of the dipeptide was coupled to the amino groups of thetetanus toxoid, whereafter the peptide of Structure (XIV) was coupled tothe tetanus toxoid via the remaining amino groups using the sameprocedure as in Example XXXII. The resultant conjugate contained fivemuramyl dipeptides and 31 peptides of Structure (XIV) per 10⁵ daltons ofcarrier respectively.

A total of eight different adjuvants were tested. The first five ofthese adjuvants were synthetic muramyl dipeptidehydrophilic analoguesobtained from Ciba-Geigy Ltd., these five dipeptides being:

(a) CGP 11637, of the formula NAc-nor Mur-L. Ala-D. iso Gln;

(b) CGP 14767, of formula NAc-nor-Mur-L-Abu-D.isoGln;

(c) CGP 18177 of formula NAc-Mur (6-0-stearoyl)-L.Ala-D.isoGln;

(d) CGP 18741 of formula NAc-nor-Mur (6-0-stearoyl)-L.Ala-D.isoGln; (e)CGP 19835 of formula NAc-Mur-L.Ala-D.isoGln-L.Ala-Cephalin.

The sixth adjuvant was another muramyl dipeptide obtained from SyntexCorporation, Palo Alto, Calif. being:

(f) DT-1, of formula NGlycol-Mur-L. a -Abu-D.isoGln.

The last two adjuvants were lipophilic adjuvants manufactured byCiba-Geigy Ltd., as follows:

(g) CGP 16940, of formula N-Palmitoyl-S-2(R,S)-3-dipalmitoyloxy-propyl!L-Cys-L-Ser-L-Ser-L-Asn-L-Ala-L-Glu; and

(h) CGP 12908, a highly purified lipoprotein from the cell membranes ofE. Coli B.

The vehicles used in these experiments were:

(a) an aqueous solution of 0.01M sodium phosphate and 0.14M sodiumchloride, of pH 7.0, hereinafter designated BPS;

(b) Incomplete Freunds' adjuvant comprising 1.5 parts by volume ofArlacel A (mannide monooleate) and 8.5 parts by volume of Klearol, bothreagents being obtained from the same sources as in Example XXXII, theadjuvant being referred to hereinafter as IFA;

(c) Squalene-Arlacel A, comprising four parts by volume Squalene(obtained from Sigma Chemicals, St. Louis, Mo. and one part by volumeArlacel A;

(d) Squalane-Arlacel A, comprising four parts by volume Squalane(obtained from Eastman Kodak, Rochester, N.Y. and one part by volumeArlacel A;

(e) Peanut oil adjuvant, comprising 10 parts by volume peanut oil(obtained from Merck, Munich, West Germany) and one part by volume egglecithin;

(f) Liposomes adjuvant, comprising 12 parts by weight egg lecithin and1.6 parts by weight cholesterol; and

(g) Alum adjuvant, comprising 10 percent by weight potassium alumprecipitated with 1N sodium hydroxide, as described in M. W. Chase andC. A. Williams (eds.), "Methods in Immunology and Immunochemistry", Vol.I, Preparation of Antigens and Antibodies, Academic Press, New York(1967), pp. 201-202.

The experimental animals used in these studies were the four inbredstrains of mice C3H/He, C57BL/6, DBA/1 and SJL, obtained from the samesource as in Example XXXII. The mice were retired breeders of more than32 weeks of age and weighed 25-30 grams. Also used were geneticallyheterogenous New Zealand White rabbits weighing 2-4 kg. obtained fromthe same source. The mice were immunized subcutaneously with a primaryimmunization at day 0 and boosters at 21 and 38 days and, in someexperiments, at day 55. Unless otherwise stated, each injectioncomprised 200 mg. of conjugate and 100 mg. of adjuvant. Blood sampleswere collected from the mice 28, 35, 45, 52, 62, and 69 days after theprimary immunization.

The rabbits were immunized intramuscularly three times at 21 dayintervals, each injection comprising 500 mg. of conjugate and 500 μg. ofadjuvant unless otherwise stated. Blood samples from the rabbits werecollected weekly on the 21st day after the first immunization. In thecase of the blood samples from both the mice and the rabbits, the serumwas separated from the remaining components of the blood and stored at-20° C. prior to analysis. Complete Freund's adjuvant purchased orprepared as in Example XXXII above was used as a reference adjuvant. Thelevels of antibody in the blood sera reacting to the peptide in theconjugate or HCG were measured using the same double-antibody techniqueas in Example XXXII and the test results were evaluated using the sameMann-Whitney U-test as in that Example. In experiments in which antigenbinding levels were pooled within each experimental group, Chi-squaredanalysis was used to determine significant differences.

RESULTS

In a first series of tests, various adjuvants were evaluated using IFAas the vehicle. The Structure (XII)/tetanus toxoid conjugate wascombined separately with the adjuvants CGP 11637, CGP 14767, CGP 12908and CGP 16940. The resultant conjugate/adjuvant mixtures wereincorporated into IFA emulsions and a parallel series of emulsions wereprepared using the conjugate (without any adjuvant) in Complete Freunds'Adjuvant (CFA). Each separate emulsions was administered to four groupseach comprising five mice from one of the four inbred strains. Antibodylevels to Structure (XII) and HCG were determined 52 days after theprimary immunization and the results are shown in Table 14 below.

                                      TABLE 14                                    __________________________________________________________________________                 Adjuvant                                                                Mouse CFA (Control)                                                                          CGP 11637                                                                              CGP 14767                                                                              CGP 12908                                                                              CGP 16940                    Antigen                                                                              Strain                                                                              ng/ml                                                                             (sd) ng/ml                                                                             (sd) ng/ml                                                                             (sd) ng/ml                                                                             (sd) ng/ml                                                                             (sd)                     __________________________________________________________________________    Structure (XII)                                                                      C57BL/6                                                                             117.7                                                                             (59.5)                                                                             70.9                                                                              (34.3)                                                                             53.0                                                                              (62.9)                                                                             281.7                                                                             (237.4)                                                                            110.0                                                                             (76.9)                          DBA/1 411.6                                                                             (268.2)                                                                            42.5                                                                              (9.6)                                                                              22.2                                                                              (14.5)                                                                             528.3                                                                             (342.5)                                                                            173.0                                                                             (37.4)                          C3H/He                                                                              566.8                                                                             (169.9)                                                                            84.0                                                                              (26.3)                                                                             74.9                                                                              (7.4)                                                                              66.4                                                                              (*)  19.2                                                                              (11.6)                          SJL   379.2                                                                             (330.1)                                                                            60.1                                                                              (44.7)                                                                             91.6                                                                              (57.9)                                                                             415.3                                                                             (254.7)                                                                            369.0                                                                             (319.7)                  CG     C57BL/6                                                                             20.4                                                                              (6.1)                                                                              18.3                                                                              (8.0)                                                                              21.0                                                                              (5.8)                                                                              47.2                                                                               (52.8)                                                                            1.0 (1.2)                           DBA   10.0                                                                              (4.0)                                                                              10.6                                                                              (7.4)                                                                              11.1                                                                              (12.2)                                                                             45.2                                                                               (73.7)                                                                            <1.0                                                                              (0.0)                           C3H/He                                                                              57.9                                                                              (47.9)                                                                             21.5                                                                              (27.9)                                                                             21.4                                                                              (12.4)                                                                             31.6                                                                              (*)  <1.0                                                                              (0.0)                           SJL   253.7                                                                             (413.0)                                                                            13.8                                                                              (12.9)                                                                             5.4 (0.5)                                                                              45.9                                                                               (39.1)                                                                            8.6 (7.4)                    __________________________________________________________________________     (*)One mouse only (n = 5)                                                

The data in Table 14 reveal significant differences among the fourstrains of mice immunized with CFA. The differences between the levelsof antibody to Structure (XII) between the DBA/1 and C3H/He groups onthe one hand and the C57BL/6 group on the other are significant at thep=0.05 level. Moreover, the differences between the HCG antibody levelsof the SJL group on the one hand and the DBA/1 and the C57BL/6 group onthe other are also significant at the p=0.05 level.

Table 15 below presents statistical comparisons of antibody levelsproduced in mice receiving each of the four adjuvants in comparison withthe antibody levels in mice receiving the CFA.

                                      TABLE 15                                    __________________________________________________________________________    Adjuvant (Compared with CFA)                                                                 CGP 11637                                                                            CGP 14767                                                                            CGP 12908                                                                            CGP 16940                                 Antigen Mouse Strain                                                                         p*     p*     p*     p*                                        __________________________________________________________________________    Structure (XII)                                                                       C57BL/6                                                                              0.22-0.31                                                                            0.096  0.420  1.0-1.158                                         DBA/1  0.008  0.008  0.556  0.016                                             C3H/He 0.016  0.134  0.400  0.028                                             SJL    0.032  0.072  0.904  1.000                                     HCG     C57BL/6                                                                              1.000  1.000  0.690  0.008                                             DBA/1  0.904  0.548  0.904  0.016                                             C3H/He 0.200  0.534  1.200  0.028                                             SJL    0.032  0.036  0.280  0.036                                     __________________________________________________________________________     *Numbers underlined are significant                                      

Of the four adjuvants tested, only the lipophilic adjuvant CGP 12908induced responses in all four strains of mice that were notsignificantly different from those of the respective CFA groups, asregards antibody levels to either HCG or Structure (XII). The otherlipophilic adjuvant CGP 16940, produced significantly lower levels ofantibody to Structure (XII) in the mouse strains DBA/1 and C3H/He, andsignificantly lower levels of antibody to HCG in all four mouse strains.The hydrophilic adjuvant CGP 14767 produced Structure (XII) antibodylevels significantly lower than those produced by CFA in only one mousegroup, the strain DBA/1, while the Structure (XII) antibody levelsproduced by the other hydrophilic adjuvant CGP 11637 were significantlylower in the three mouse groups DBA/1, C3H/He and SJL than in thecorresponding CFA groups. The HCG antibody levels produced by both thehydrophilic adjuvants CCG 11637 and 14767 were not significantlydifferent from those of the corresponding CFA groups, only the SJLstrain producing significantly different results in both cases.

Since the responses of the mice to immunization with the Structure(XII)/tetanus toxoid conjugate were more dependent upon the adjuvantused than the strain of mouse injected, genetic differences among thevarious strains were difficult to assess. However, the C57BL/6 strainmice did not show any significant differences in Structure (XII)antibody levels with any of the four adjuvants tested, as compared withthe CFA immunized mice. On the other hand, mice of the DBA/1 strain didshow significantly lower Structure (XII) antibody levels with three ofthe four adjuvants tested, as compared with the CFA immunized mice.Accordingly, in some of the later experiments only these two strains ofmice were used for assessing genetic differences.

A parallel series of tests using the same four adjuvants, as well as thehydrophilic adjuvant DT-1 in conjunction with Structure (XII)/tetanustoxoid conjugate and IFA, produced somewhat different results, as shownin FIG. 7, which shows the mean HCG antibody levels averaged over thegroups of four rabbits used, from 3-13 weeks after primary immunization.As with the mouse tests, a group of four rabbits was immunized with theconjugate incorporated into CFA for comparison. FIG. 7 shows that theHCG antibody levels in all groups were substantially constant from the9-13 weeks after primary immunization, and therefore statisticalevaluation of the antibody levels was conducted after pooling datawithin each group during this five week period.

As compared with the CFA immunized rabbits, only the rabbits receivingthe adjuvant CGP 16940 produced significantly lower HCG antibody levels(p<0.05). The rabbits receiving adjuvants CGP 11637 and DT-1 producedHCG antibody levels significantly greater than the rabbits receivingadjuvants CGP 16940 and 12908 (p<0.05), but not significantly higherthan the rabbits receiving adjuvant CGP 14767 or the rabbits receivingCFA.

Further tests were conducted in mice and rabbits to determine theeffects of the various vehicles on antibody levels. In the mice tests,groups of five mice from each of the strains C57BL/6 and DBA/1 wereimmunized with the conjugate of tetanus toxoid and Structure (XIV) (the111-145 sequence of β-HCG with the N-terminal spacer sequence) inconjunction with one of the vehicles PBS, IFA, liposomes andSqualene/Arlacel. No adjuvants were used in immunizing the micereceiving the vehicles, but a group of mice from each strain wereimmunized with the conjugate incorporated into CFA for comparisonpurposes.

The mean Structure (XIV) and HCG antibody levels for each group of mice35 and 52 days after the primary immunization are shown in Table 16 asbetween the CFA Control group with the groups receiving the variousother vehicles by the aforementioned U-test are shown in Table 17 below.Table 17 shows that the Structure (XIV) antibody levels in the DBA/1mice were significantly higher than those in the C57BL/6 mice at either35 or 52 days after the primary immunization. In terms of HCG antibodylevels the DBA/1 mice were significantly lower than those of the C57BL/6mice 35 days after immunization, but not significantly lower 52 daysafter primary immunization.

The U-test factors in Table 17 show that, in the C57BL/6 mice groups 35days after primary immunization, only the group receiving the IFA didnot have significantly lower levels of antibodies to both Structure(XIV) and HCG than those of the mice receiving CFA. However, the results52 days after primary immunization are strikingly different: nosignificant differences between Structure (XIV) or HCG antibody levelsexisted at that time between the mice receiving CFA and those receivingBPS, IFA or liposomes vehicles. Indeed, the Squalene/Arlacel vehicleimmunized mice had levels of antibodies to both Structures (XIV) and HCGwhich were significantly higher than those of any other vehicleincluding CFA. The responses of the DBA/1 mice were not similar to thoseof the C57BL/6 mice. Thirty-five days after primary immunization, thefour groups of DBA/1 mice immunized with the vehicles under test hadStructure (XIV) antibody levels significantly greater than those of themice receiving CFA, but only the group of mice receiving liposomesvehicle had HCG antibody levels significantly lower than the CFA group.In all the groups of DBA/1 mice 52 days after primary immunization, theantibody levels to both Structure (XIV) and HCG were not significantlydifferent from the CFA group.

                                      TABLE 16                                    __________________________________________________________________________                    Sera 35 days after Primary                                                                      Sera 52 days after Primary                                  Immunization      Immunization                                                Antibody Level M/L × 10.sup.10 (sd)                                                       Antibody Level M/L × 10.sup.10                                          (sd)                                        Mouse Strain                                                                         Vehicle  Structure (XIV)                                                                        HCG      Structure (XIV)                                                                        HCG                                __________________________________________________________________________    C57BL/6                                                                              PBS      3.2 (2.7)                                                                              2.5 (1.2)                                                                              3.1 (2.7)                                                                              2.8 (1.8)                                 IFA      116.8                                                                             (90.4)                                                                             107.9                                                                             (77.6)                                                                             24.3                                                                              (11.4)                                                                             15.1                                                                              (10.8)                                Liposomes                                                                              2.0 (0.0)                                                                              2.0 (0.0)                                                                              13.5                                                                              (9.6)                                                                              12.4                                                                              (8.3)                                 Squalene/Arlacel                                                                       22.5                                                                              (28.4)                                                                             9.1 (10.0)                                                                             397.1                                                                             (149.2)                                                                            331.4                                                                             (153.3)                               CFA (Control)                                                                          184.5                                                                             (107.6)                                                                            141.6                                                                             (70.5)                                                                             21.6                                                                              (25.9)                                                                             12.5                                                                              (11.6)                         DBA/1  PBS      54.0                                                                              (31.8)                                                                             16.1                                                                              (8.6)                                                                              94.4                                                                              (61.8)                                                                             19.1                                                                              (14.5)                                IFA      203.1                                                                             (174.3)                                                                            25.6                                                                              (37.4)                                                                             240.2                                                                             (137.5)                                                                            21.1                                                                              (22.2)                                Liposomes                                                                              2.1 (0.3)                                                                              2.0 (0.0)                                                                              74.0                                                                              (136.6)                                                                            17.6                                                                              (21.3)                                Squalene/Arlacel                                                                       23.0                                                                              (8.6)                                                                              13.6                                                                              (5.9)                                                                              205.2                                                                             (97.2)                                                                             11.8                                                                              (10.0)                                CFA (Control)                                                                          442.1                                                                             (74.2)                                                                             40.0                                                                              (23.1)                                                                             206.1                                                                             (130.5)                                                                            14.2                                                                              (5.6)                          __________________________________________________________________________

                                      TABLE 17                                    __________________________________________________________________________                 Sera 35 days after                                                                      Sera 52 days after                                          CFA     Primary Immunization                                                                    Primary Immunization                                   Mouse                                                                              Compared                                                                              U-test (p*) for                                                                         U-test (p*) for                                        Strain                                                                             With    Structure (XIV)                                                                      HCG                                                                              Structure (XIV)                                                                      HCG                                             __________________________________________________________________________    C57BL/6                                                                            PBS     0.004  0.004                                                                            0.178  0.052-0.082                                          IFA     0.246  0.246                                                                            0.330  0.930-1.070                                          Liposomes                                                                             0.008  0.008                                                                            0.842-1.000                                                                          0.842-1.000                                          Squalene/Arlacel                                                                      0.008  0.008                                                                            0.016  0.016                                           DBA/1                                                                              PBS     0.016  0.112                                                                            0.286  0.904                                                IFA     0.032  0.310                                                                            1.000  0.842                                                Liposomes                                                                             0.008  0.008                                                                            0.310  0.548                                                Squalene/Arlacel                                                                      0.008  0.056                                                                            1.096  0.556                                           __________________________________________________________________________     *Numbers underlined are significant at p = 0.05 level                    

A similar series of tests were run in rabbits using the four vehiclesSqualane/Arlacel A, Squalene/Arlacel B, peanut oil and alum. These testswere conducted using the same Structure (XIV)/tetanus toxoid conjugate,but the adjuvant DT-1 was used. Again, a control group of rabbits wereimmunized with the conjugate emulsified in CFA without a vehicle.Antibody levels to HCG were measured throughout the 13 week immunizationperiod and the data obtained from each group of rabbits at the week ofpeak antibody level were pooled with the values obtained from the samegroup one week earlier and one week later. The results are shown inTable 18 below. The HCG antibody levels in rabbits receivingSqualene/Arlacel were significantly higher than those of all othergroups, including the rabbits receiving CFA. No significant differencesin HCG antibody levels existed between the rabbits receivingSqualane/Arlacel and those receiving CFA, while the rabbits receivingpeanut oil or alum vehicles had significantly lower HCG antibody levelsthan those receiving CFA.

The efficacy of the Squalene/Arlacel A vehicle in increasing theantibody levels in the injected animals is surprising, especially sinceSqualane/Arlacel has not previously been used as a vehicle in a vaccine,although Squalene is used in topical preparations such as ointments andcosmetics. Although the Squalane/Arlacel A vehicle was not as effectiveas the Squalene/Arlacel vehicle, it was as efficacious as CFA. Both theSqualene/Arlacel and the Squalane/Arlacel vehicles should be clinicallyacceptable for use in human beings, since they appear to produce littleor no irritation at the sight of injection, whereas CFA is known not tobe clinically acceptable for use in human beings since it tends toproduce intense irritation, abscesses, etc. at the point of injection.

                                      TABLE 18                                    __________________________________________________________________________             Antibody Level M/L × 10.sup.10                                 Vehicle  n Minimum                                                                            Median                                                                            Maximum                                                                            Mean                                                                             95% C.I.                                                                            sd p*                                       __________________________________________________________________________    CFA (Control)                                                                          12                                                                              52.4 150.8                                                                             320.2                                                                              161.3                                                                            101.7-220.9                                                                         93.7                                        Squalene/Arlacel A                                                                     11                                                                              189.0                                                                              312.6                                                                             833.7                                                                              400.1                                                                            225.7-574.4                                                                         226.7                                                                            0.01                                     Squalane/Arlacel A                                                                     11                                                                              44.2 150.6                                                                             290.0                                                                              168.5                                                                            114.5-222.5                                                                         80.4                                                                             NS                                       Peanut Oil                                                                             9 12.5 22.5                                                                              27.1 21.3                                                                             17.5-25.1                                                                           4.9                                                                              <0.001                                   Alum Precipitate                                                                       12                                                                              5.8  8.4 16.1 9.9                                                                               7.9-11.9                                                                           3.2                                                                              <0.001                                   __________________________________________________________________________     *Probability, compared with CFA. NS = not significant (p ≧ 0.05)  

To evaluate simultaneously combinations of various adjuvants and variousvehicles, and thus to detect any second order effects due to theinteractions of particular adjuvants with particular vehicles, groups offive or six mice from each of the strains C57BL/6 and DBA/1 wereimmunized with preparations comprising the same Structure (XIV)/tetanustoxoid conjugate used in the preceding tests in combination with one ofthe synthetic adjuvants CGP 18177, 18741 or 19834 and one of the threevehicles Squalene/Arlacel, liposomes and peanut oil. As in the precedingtest described above with reference to Tables 16 and 17, HCG antibodylevels were determined in sera collected 35 and 52 days after primaryimmunization, and the results are shown in Table 19 below. The data inTable 19 reveal no significant differences between the levels ofantibodies in the groups of mice injected with the same adjuvant indifferent vehicles, nor between the groups receiving the same vehicleand different adjuvants, at either 35 or 52 days after primaryimmunization (i.e. p>0.05 in all cases). Furthermore, no differencessignificant at the p=0.05 level were detected between the levels betweencorresponding groups of mice of difference strains.

Table 20 below shows data similar to those in Table 19 but relating toStructure (XIV) antibody levels instead of HCG antibody levels. Analysisof the date in Table 20 shows that 35 days after primary immunization inthe C57BL/6 mice, mice receiving the adjuvant CGP 18177 producedsignificantly higher levels of antibodies than the group receivingadjuvant CGP 19835 (p<0.05). However, the similar differences at 52 daysafter primary immunization were not significant at the p=0.05 level.Comparison of the three adjuvants of the DBA/1 mice 35 days afterprimary immunization shows no significant differences between theadjuvants in Squalene/Arlacel or peanut oil vehicles but the Structure(XIV) antibody levels produced by CGP 18177 in liposomes vehicle weresignificantly higher than those produced by CGP 19835 in the samevehicle (p<0.05). The results 52 days after primary immunization showthe same pattern of significant differences as the results 35 days afterprimary immunization.

While mean HCG antibody levels were generally higher in mice immunizedwith Squalene/Arlacel vehicle and Structure (XIV) antibody levels wereconsistently higher using this vehicle, regardless of adjuvant or mousestrain, these differences were not significant in view of thevariability in the responses of the mice. Although no significantdifferences were found between the two strains of mice as regards to HCGantibody levels, the DBA/1 mice produced higher mean Structure (XIV)antibody levels in all vehicle and adjuvant groups than the C57BL/6 micealthough the differences were not statistically different.

                                      TABLE 19                                    __________________________________________________________________________                  Sera 35 days after Primary                                                                   Sera 52 days after Primary                                     Immunization   Immunization                                                   Antibody Level M/L × 10.sup.-10 (sd)                                                   Antibody Level M/L × 10.sup.-10 (sd)                     in             in                                               Adjuvant                                                                            Vehicle C57BL/6                                                                              DBA/1   C57BL/6                                                                              DBA/1                                     __________________________________________________________________________    CGP 18177                                                                           Squalene/Arlacel                                                                      17.7                                                                             (9.4)                                                                             9.6 (14.6)                                                                            10.2                                                                             (4.9)                                                                             12.3                                                                              (12.8)                                      Liposomes                                                                             14.8                                                                             (4.9)                                                                             10.1                                                                              (1.7)                                                                             16.7                                                                             (5.4)                                                                             15.5                                                                              (8.3)                                       Peanut Oil                                                                            9.1                                                                              (8.8)                                                                             15.2                                                                              (6.4)                                                                             13.3                                                                             (14.8)                                                                            7.1 (2.1)                                 CGP 18741                                                                           Squalene/Arlacel                                                                      13.7                                                                             (6.5)                                                                             10.5                                                                              (8.4)                                                                             7.8                                                                              (3.9)                                                                             9.1 (8.0)                                       Liposomes                                                                             9.6                                                                              (6.5)                                                                             8.8 (5.1)                                                                             5.6                                                                              (6.6)                                                                             13.8                                                                              (5.2)                                       Peanut Oil                                                                            23.9                                                                             (8.4)                                                                             13.9                                                                              (9.3)                                                                             4.6                                                                              (3.9)                                                                             10.5                                                                              (6.9)                                 CGP 19835                                                                           Squalene/Arlacel                                                                      13.5                                                                             (6.6)                                                                             13.2                                                                              (8.9)                                                                             10.4                                                                             (7.1)                                                                             16.8                                                                              (3.2)                                       Liposomes                                                                             7.4                                                                              (9.4)                                                                             16.7                                                                              (14.6)                                                                            2.0                                                                              (0.0)                                                                             15.2                                                                              (9.8)                                       Peanut Oil                                                                            10.7                                                                             (7.4)                                                                             11.2                                                                              (5.4)                                                                             2.7                                                                              (1.5)                                                                             5.7 (*)                                   __________________________________________________________________________     (*)One mouse only from n = 5                                             

                                      TABLE 20                                    __________________________________________________________________________                  Sera 35 days after Primary                                                                   Sera 52 days after Primary                                     Immunization   Immunization                                                   Antibody Level M/L × 10.sup.-10 (sd)                                                   Antibody Level M/L × 10.sup.-10 (sd)                     in             in                                               Adjuvant                                                                            Vehicle C57BL/6                                                                              DBA/1   C57BL/6                                                                              DBA/1                                     __________________________________________________________________________    CGP 18177                                                                           Squalene/Arlacel                                                                      50.1                                                                             (43.1)                                                                            160.7                                                                             (120.1)                                                                           163.7                                                                            (100.4)                                                                           419.4                                                                             (345.4)                                     Liposomes                                                                             32.2                                                                             (9.4)                                                                             104.0                                                                             (35.4)                                                                            35.0                                                                             (21.5)                                                                            206.4                                                                             (3.5)                                       Peanut Oil                                                                            16.8                                                                             (13.3)                                                                            69.4                                                                              (36.5)                                                                            92.6                                                                             (106.0)                                                                           91.4                                                                              (59.2)                                CGP 18741                                                                           Squalene/Arlacel                                                                      21.4                                                                             (10.9)                                                                            130.4                                                                             (115.5)                                                                           65.8                                                                             (42.9)                                                                            371.7                                                                             (364.7)                                     Liposomes                                                                             10.9                                                                             (11.3)                                                                            54.0                                                                              (28.4)                                                                            30.3                                                                             (40.3)                                                                            134.5                                                                             (62.0)                                      Peanut Oil                                                                            2.0                                                                              (0.0)                                                                             89.4                                                                              (127.0)                                                                           27.1                                                                             (38.7)                                                                            46.2                                                                              (31.4)                                CGP 19835                                                                           Squalene/Arlacel                                                                      18.8                                                                             (6.6)                                                                             200.2                                                                             (201.8)                                                                           95.4                                                                             (70.5)                                                                            195.9                                                                             (177.3)                                     Liposomes                                                                             6.0                                                                              (8.9)                                                                             19.5                                                                              (13.2)                                                                            44.5                                                                             (36.4)                                                                            92.7                                                                              (38.4)                                      Peanut Oil                                                                            2.0                                                                              (0.0)                                                                             148.1                                                                             (141.7)                                                                           45.4                                                                             (57.5)                                                                            7.2 (*)                                   __________________________________________________________________________     (*)One mouse from n = 5                                                  

A similar series of tests were then carried out in rabbits using thesame Structure (XIV)/tetanus toxoid conjugate, the adjuvants CGP 18177,18741, and 19835 tested in mice, plus the additional adjuvants CGP 11637and DT/1, using groups of four rabbits and Squalene/Arlacel as thevehicle. Again, a control group of rabbits were immunized with theconjugate incorporated into CFA without a vehicle. The mean HCG antibodylevels in sera collected from the various groups of rabbits weekly from3-12 weeks after primary immunization are shown in Table 21 below,together with the corresponding standard deviations. The data in Table21 show that the highest antibody levels were achieved in rabbitsreceiving the adjuvants CGP 11637 and DT/1 10 weeks after primaryimmunization. Comparison of the antibody levels produced by theseadjuvants with the control group receiving CFA showed that the increasesin antibody levels with both adjuvants were significant at the p=0.05level. Adjuvant CGP 18177 also produced higher antibody levels than CFA,the difference being significant at the p=0.05 level, and thedifferences between the antibody levels produced by the three adjuvantsCGP 11637, DT/1 and CGP 18177 are not significant at the p=0.05 level.The adjuvants CGP 19835 and 18741 produced antibody levels which werelower than the CFA rabbits, the difference being significant at thep=0.05 level.

                                      TABLE 21                                    __________________________________________________________________________    Weeks After   Mean Antibody Level-.sup.125 I-HCG M/L × 10.sup.10                      (sd)                                                            Primary       Adjuvant                                                        Immunization                                                                         CFA (Control)                                                                        CGP 11637                                                                            CGP 19835                                                                            CGP 18741                                                                            CGP 18177                                                                            DT-1                                __________________________________________________________________________     3*    14.3                                                                             (8.6)                                                                             16.3                                                                             (11.8)                                                                            19.3                                                                             (8.8)                                                                             5.9                                                                              (1.4)                                                                             19.9                                                                             (19.2)                                                                            31.1                                                                             (8.4)                            4      69.0                                                                             (26.8)                                                                            177.8                                                                            (105.0)                                                                           286.7                                                                            (113.8)                                                                           9.8                                                                              (4.8)                                                                             215.1                                                                            (207.6)                                                                           145.1                                                                            (56.6)                           5      158.1                                                                            (88.8)                                                                            211.9                                                                            (105.2)                                                                           140.3                                                                            (82.2)                                                                            6.2                                                                              (0.6)                                                                             204.6                                                                            (137.6)                                                                           184.1                                                                            (54.2)                            6*    150.4                                                                            (66.0)                                                                            297.2                                                                            (152.4)                                                                           172.0                                                                            (74.4)                                                                            8.9                                                                              (7.4)                                                                             218.2                                                                            (46.2)                                                                            268.6                                                                            (170.4)                          7      171.2                                                                            (193.4)                                                                           381.2                                                                            (228.8)                                                                           123.4                                                                            (46.6)                                                                            81.1                                                                             (46.6)                                                                            441.9                                                                            (144.2)                                                                           371.5                                                                            (183.8)                          8      185.2                                                                            (200.4)                                                                           418.6                                                                            (292.4)                                                                           138.9                                                                            (39.2)                                                                            75.4                                                                             (64.4)                                                                            544.9                                                                            (226.6)                                                                           366.2                                                                            (229.2)                          9      238.6                                                                            (143.4)                                                                           480.0                                                                            (260.6)                                                                           105.2                                                                            (15.4)                                                                            74.9                                                                             (4S.6)                                                                            456.2                                                                            (171.0)                                                                           654.8                                                                            (380.6)                          10     324.5                                                                            (244.0)                                                                           708.6                                                                            (329.0)                                                                           151.3                                                                            (38.4)                                                                            121.8                                                                            (51.4)                                                                            631.0                                                                            (377.8)                                                                           729.4                                                                            (455.4)                          11     210.8                                                                            (140.0)                                                                           628.5                                                                            (445.8)                                                                           169.3                                                                            (40.0)                                                                            156.8                                                                            (25.0)                                                                            584.0                                                                            (213.4)                                                                           627.5                                                                            (340.8)                          12     195.3                                                                            (139.0)                                                                           398.5                                                                            (350.4)                                                                           107.8                                                                            (20.2)                                                                            97.6                                                                             (10.6)                                                                            270.0                                                                            (175.2)                                                                           559.0                                                                            (460.4)                          __________________________________________________________________________     *Booster immunization given at this time                                 

A final series of tests were run in mice to determine the effect ofcoupling one of the adjuvants to the peptide/tetanus toxoid conjugateinstead of merely administering the peptide/tetanus toxoid conjugatemixed with the adjuvant. Groups of five mice from each of the strainsC57BL/6 and DBA/1 were immunized with the Structure (XIV)/tetanustoxoid/CGP 11637 conjugate described above, separate groups of micebeing immunized with the conjugate in association with each of thevehicles PBS, IFA, liposomes and Squalene/Arlacel. As before, a Controlgroup from each strain was immunized with the conjugate incorporatedinto CFA for comparison purposes.

FIG. 8 shows the mean HCG and Structure (XIV) antibody levels in themice as a function of time after primary immunization (the coding forthe various vehicles in FIG. 8 is the same as that in FIG. 7). FIG. 8ashows the HCG antibody levels of the C57BL/6 mice. The elevated antibodylevels produced by CFA and IFA 35 days after primary immunization arenot significant at the p=0.05 level and were not sustained after thebooster immunizations at 33 and 55 days. The mice receiving theconjugate in Squalene/Arlacel vehicles did not respond significantlyuntil the third bleeding, 45 days after primary immunization, but thereHCG antibody levels increased progressively thereafter and were higherby an amount significant at the p=0.05 level as compared with those ofthe other mouse groups. The Structure (XV) antibody levels of the samemice shown in FIG. 8b parallel those of the HCG antibody levels andstatistical analysis shows that the same differences are significant.

The HCG antibody levels of the DBA/1 mice shown in FIG. 8c areconsiderably lower than those of the corresponding C57BL/6 mice.Fifty-two days after primary immunization, the HCG antibody levels inthe DBA/1 mice receiving the Squalene/Arlacel vehicle were notsignificantly different from those of the CFA Control group (p>0.05),but were significantly higher than the levels achieved in the micereceiving the other vehicles (p<0.0). Sixty-nine days after primaryimmunization, there were no differences significant at the p=0.05 levelbetween any of the groups of DBA/1 mice. However, as shown in FIG. 8d,unlike the results obtained with the C57BL/6 mice, in the DBA/1 mice,the antibody levels to Structure (XIV) do not follow the antibody levelsto HCG. The Structure (XIV) antibody levels in the DBA/1 mice receivingCFA or IFA were elevated 28 days after the primary immunization andthereafter declined until day 52. Later, the CFA immunized mice levelsrose in response to the booster injection administered at day 55, whilethe antibody levels in the IFA mice continued to fall. No differencessignificant at the p=0.05 level were found between the antibody levelsin the mice receiving CFA, IFA or Squalene/Arlacel 52 days after theprimary immunization, but at this time the differences between the micereceiving CFA and those receiving PBS and liposomes vehicles weresignificant at the p=0.05 level. After 69 days from primaryimmunization, while the difference between the CFA groups and theremaining vehicles were significant at the p=0.05 level, the differencesbetween the other groups were not significant at this level.

The experimental results described above clearly demonstrate theefficacy of some of the synthetic adjuvants in increasing antibodyproduction when administered in conjunction with the modifiedpolypeptide of the invention. Some of the adjuvants, in particular CGP11637 and 18177 and DT/1, when administered in certain vehicles,especially IFA and Squalene/Arlacel produced antibodies exceeding thoseproduced by CFA. In general, the hydrophilic muramyl peptide adjuvantswere superior to the lipophilic adjuvants in enhancing the antibodyproduction caused by the hydrophilic Structure (XIV)/tetanus toxoidconjugate. The experimental results also show that the delivery systemused to administer the antigen and adjuvant is of critical importance inproducing an enhanced response to the conjugate. For example, theantibody responses of the C57BL/6 mice which received the conjugate inSqualene/Arlacel without adjuvant were greater than those of the micereceiving the conjugate in CFA. Adding an adjuvant to this vehicleproduced only a slight increase in the antibody levels. Theseobservations are confirmed by those in rabbits receiving the conjugateand the synthetic adjuvant in different vehicles, and differentadjuvants in the same vehicle. Squalane/Arlacel and peanut oil emulsionswere efficacious, though less effective than Squalene/Arlacel.

To sum up, based upon all the foregoing data it appears that the optimumvehicle was Squalene/Arlacel, which was superior to the others in almostevery formulation and that the best adjuvants are CGB 11637 and DT/1,which were more efficacious in various vehicles than other adjuvantstested simultaneously. Squalane/Arlacel was also a highly acceptablevehicle although somewhat less efficacious than Squalene/Arlacel.

EXAMPLE XXXIV

This example illustrates a use of a modified polypeptide of theinvention in repressing a carcinoma which produces a chorionicgonadotropin-like material. This material by John A. Kelen, Arnost Kolinand Hernan F. Acevedo is in press and will shortly appear in Cancer.

The rat mammary adenocarcinoma R 3230 AC has been shown, usingimmunocytochemical, radioimmunoassay (RIA) and bioassay methods intissue sections and cell cultures to product a chorionic gonadotropin(CG)-like material; however this CG-like material cannot be detected inthe sera of the animals bearing the carcinoma. The detection of theCG-like material is described in:

(a) Malkin et al., "Immunohistochemical Detection of Ectopic Hormones inExperimental Rat Tumors", in F. G. Lehmann (ed.), Carcino-embryonicProteins, Vol. 2, Elsevier-North Holland Biomedical Press, New York,Amsterdam (1979), pp. 751-758.

This adenocarcinoma can be propagated in cell culture and its cellsretain their morphology and malignant characteristics. Intravenousinjection of these cells into isologous Fischer 344 rats gives rise tonumerous foci of this neoplasm in the lungs within 8-10 days, theanimals then becoming very sick and then dying within 12-15 days.

The R 3230 adenocarcinoma (obtained from Dr. A. Bogden, Mason ResearchInstitute, Worcester, Mass.) was cultured from explants of subcutaneoustumors grown for 20-21 days in Fischer 344 female rats. The cultureswere maintained in RPMI 1640 medium supplemented with 10% fetal coughserum. Upon reaching confluency, the cells were dispersed by means oftrypsin-EDTA (1:250) and passaged five times. During the earlystationary growth phase, the cultured cells were dispersed for in vivoadministration. The cell density was determined using a hemocytometerand viability was tested by the Trypan blue exclusion method. Onemillion cells were then injected into the tail vein of each of theexperimental animals, which were 100 female Fischer 344 rats weighing130-150

g. The animals were divided into two matched control groups of 15animals each and a test group of 70 animals. They were individuallycaged, fed Purina (Registered Trade Mark) chow and tap water ad libitum.All animals except six from the test group were sacrificed at intervalsby cervical dislocation for necropsy studies. The lungs, livers, spleenand kidneys were examined and processed for paraffin sections and H&Estaining in order to detect neoplastic foci, the diameter of the focibeing measured with an eye piece micrometer. All animals were also bledbefore sacrifice and the serum tested for the presence of antibodies toCG.

The test group of 70 rats received, prior to the injection of carcinomacells, a β-ECG/tetanus toxoid modified polypeptide of the invention.This β-HCG conjugate, and its method of preparation, is described inU.S. Pat. No. 4,161,519 to Talwar and in the following papers written bythe same author and others:

(b) Proc. Natl. Acad. Sci. U.S.A., 73, 218-222 (1976);

(c) Contraception, 18, 19-21 (1978);

(d) Fertil. Steril., 34, 328-335 (1980).

The conjugate was administered in saline once per week for a period ofthree weeks, approximately 1.3 μg. being given at each injection and thelast injection being given two weeks before administration of thecarcinoma cells. In order to study the effect of the conjugate over anextended period, a number of animals selected at random were sacrificedfor autopsy, as follows (the day when the carcinoma cells were injectedbeing taken as day 0):

    ______________________________________                                        Day       Number of Rats Sacrificed                                           ______________________________________                                        3         2                                                                   5         5                                                                   8         5                                                                   10        36                                                                  22        10                                                                  28        4                                                                   120       2                                                                   ______________________________________                                    

One of the two control groups was administered highly purified tetanustoxoid (obtained from Connaught Laboratories, Toronto, Ontario, Canada)at a rate of 0.7 μg. per injection, following the same injectionschedule as the test group followed with the conjugate. Thus, thiscontrol group received a dose of tetanus toxoid substantially equal tothat received in the conjugate by the test animals. The second controlgroup received no treatment other than the tumor suspensions. All theanimals from the test group were sacrificed for autopsy on day 10.

Testing for the presence of HCG antibodies was performed using theECG-b-RIA kit, Quantitative Method II (manufactured by SeronoLaboratories, Braintree, Mass.). To the cold standard ECG, HCG freeserum and ¹²⁵ I-HCG, an aliquot of the rat serum was added. Afterovernight incubation, the original protocol of the kit was followed inthe subsequent procedure. If all the labeled HCG was precipitated (thusindicating a high antibody titer) the test was repeated withappropriately diluted rat serum.

All the animals in the two control groups, sacrificed 10 days afterinjection of the tumor cells, showed multiple lung foci of neoplasticcells in accordance with the normal progress of this carcinoma. None ofthe 30 animals in the control groups showed neoplasms in any otherorgans. The qualitative histological appearance of the pulmonary tumordeposits was practically identical in every animal. The neoplasm is amoderately differentiated adenocarcinoma rarely producing glandularlumina. The mitotic rates are high, but tumor necrosis and inflammatoryreactions are absent. The tumor nodules range in size from a few cellsto more than one millimeter in diameter and are uniformly distributedthroughout the lung parenchyma. Using a 2.5 X objective, from 5-10 tumornodules were present in every low power camera field in the controlledanimals. In contrast, the test animals that had received theβ-HCG/tetanus toxoid conjugate rarely displayed more than 1 to 4metastatic nodules not exceeding 0.3 mm. in diameter in the entire lungsections 8-10 days after administration of the tumor cells, so that onlya few camera fields could be found containing a single metastatic focus.Most lung parenchyma were completely free of neoplasms. The histologicalappearance of the neoplasms in the test animals was the same as that inthe controls.

In all the rats receiving the conjugate, a significant antibody titerwas found, antibody levels capable of precipitating 200 mIU of HCGstandard per milliliter of serum being consistently determined. Thistiter persisted for up to 120 days after receipt of the tumor cells andcontinued to protect at that time against formation of metastatic lungfoci after a new intravenous seeding with the tumor cells. In contrast,neither of the control groups showed any measurable HCG antibody levels,regardless of whether the rats had or had not received the tetanustoxoid.

Thus, the protective effects of immunization with the conjugate prior toinjection with the tumor cells were demonstrated by the test animalsbeing alive 20 days after injection of the tumor cells (at which time,100% mortality would be expected in unprotected animals), by the absenceof lung pathology in the animals sacrificed more than 20 days afterinjection of the tumor cells, by the long term survival (more than sixmonths) and lack of deterioration in the six test animals that were notsacrificed, and by the fertility of these six surviving test animals,several of which produced normal litters after termination of the 120day observation period. This long term survival of all the six animalsand absence of any deterioration therein is especially surprising inview of the virulence of the R 3230 A carcinoma chosen for study, sinceprevious work has indicated that no rats receiving the intravenousinjection of tumor cells received by these test animals can be expectedto survive for more than about 20 days.

Since there are no detectable levels of serum HCG associated with thiscarcinoma in rats, the absence of pathological changes in the organsinvestigated and the maintenance of the reproductive functions in thesurviving test rats suggest that the antibodies act at the level of thecell membrane, probably in a cytotoxic manner, although the aboveexperimental results are not sufficient to prove this hypothesis.

EXAMPLE XXXV

This example illustrates the use of a modified polypeptide of theinvention coupled to diphtheria toxoid in repressing fertility inbaboons.

A modified polypeptide of the invention based upon Structure (XII) abovewas prepared in exactly the same manner in Example XXXI except that thepeptide was conjugated to diphtheria toxoid instead of tetanus toxoid.Also, the resultant conjugate, again containing about 22 peptides per100,000 daltons of the toxoid, was dissolved in a solution of themuramyl dipeptide CGP 11637 used in Example XXXIII, instead of theComplete Freunds' adjuvant used in Example XXXI. The resultantconjugate/adjuvant mixture was emulsified with an equal volume of the4:1 v/v Squalene/Arlacel A vehicle used in Example XXXIII. Again, thecontrol animals received diphtheria toxoid in an amount equal to thatreceived as part of the conjugate by the test animals. All detailsregarding animal feeding, group size, animal housing, mode ofadministration of vaccine and blood testing were exactly as in ExampleXXXI.

The length of the menstrual cycles and the luteal phase progesteronelevels for both the test and control groups were measured for threemenstrual cycles before immunization and five menstrual cycles afterimmunization. The results are shown in Table 22 below (which is directlycomparable with Table 7 of Example XXXI). The data in Table 22 showsthat, as in the previous experiment, no significant differences existedbetween the lengths of menstrual cycle and luteal phase progesteronelevels of the two groups of baboons either before or after immunization.

Table 23 below shows the antibody levels to Structure (XII), baboonchorionic gonadotropin and human chorionic gonadotropin produced in thetest animals injected with the conjugate during the five menstrualcycles following immunization. As in Example XXXI, the antibody levelswere determined from serum drawn from the early luteal phase of eachmenstrual cycle. Thus, the data in Table 23 may be compared directlywith those in Table 5 of Example XXXI. Comparing Tables 5 and 23, itwill be seen that the diphtheria-toxoid-containing conjugate producedslightly lower levels of antibodies to baboon chorionic gonadotropinthan did the tetanus-toxoid-containing conjugate (though this differencedoes not appear to be significant), but rather higher levels ofantibodies to human chorionic gonadotropin (and the differences betweenthe two groups for menstrual cycles Nos. 4 and 5 would appear to besignificant).

As in Example XXXI, each of the baboons was mated during the course ofthe third menstrual cycle following immunization and also mated duringthe two subsequent menstrual cycles unless, of course, a previous matingproduced a pregnancy. The results are shown in Table 24, which isdirectly comparable with Table 8 of Example XXXI. Of the control animalsimmunized only with diphtheria toxoid, 11 out of 15 became pregnant onthe first mating, three of the remaining four became pregnant after thethird mating. Thus, a total of 20 matings produced 14 pregnancies and afertility rate of 70%. On the other hand, in the test animals immunizedwith the conjugate, none of the 15 baboons became pregnant during thefirst mating, only one of the remaining baboons became pregnant at thesecond mating, and at the third mating, another one of the 14 remainingbaboons became pregnant. Thus, a total of 44 matings produced only twopregnancies for a fertility rate of 4.6%. Although statisticalcomparisons are difficult because of the very small numbers ofpregnancies involved in the conjugate-immunized baboons, it thus appearsthat the vaccine used in this Example containing Structure(XII)/diphtheria toxoid conjugate is more effective in preventingconception in baboons than the vaccine used in Example XXXI.

A detailed immunosafety study in 48 female baboons using the samevaccine revealed no detectable alteration in serum chemistry,urinalysis, immune complex formation, auto-immunity or anyhypersensitivity reactions. Histopathological evaluation of numeroustissues from sacrifice of all animals revealed no evidence of pathologyfrom immunization. Antisera raised in these baboons failed to react withany pituitary glycoprotein hormone, thus showing that the antibodiesformed in response to the vaccine did not cross-react with LH or FSH. Nochange in endogenous baboon hormone levels was observed followinginjections of the vaccine.

                                      TABLE 22                                    __________________________________________________________________________    Length of menstrual cycles and luteal phase progesterone levels                            Menstrual Cycles                                                              Pre-Immunization                                                                       Post-Immunization                                       Baboons Immunized With                                                                     -3 -2 -1 1  2  3  4  5                                           __________________________________________________________________________    Diphtheria Toxoid                                                             Cycle Length (days)                                                           Mean         34.4                                                                             33.5                                                                             33.2                                                                             32.7                                                                             33.1                                                                             34.0                                                                             30.0                                                                             31.0                                        SE           1.0                                                                              0.9                                                                              0.6                                                                              0.7                                                                              0.8                                                                              0.7                                                                              -- --                                          n.sup.(1)    15 15 15 15 15 4  1  1                                           Progesterone (ng/ml)                                                          Mean         6.9                                                                              6.8                                                                              6.0                                                                              4.3                                                                              6.0                                                                              7.0                                                                              5.9                                                                              6.0                                         SE           0.6                                                                              0.5                                                                              0.4                                                                              0.5                                                                              0.5                                                                              0.9                                                                              -- --                                          n.sup.(1)    15 15 15 15 15 4  1  1                                           Conjugate                                                                     Cycle Length (days)                                                           Mean         33.2                                                                             32.7                                                                             34.1                                                                             33.9                                                                             32.7                                                                             32.1                                                                             32.2                                                                             32.5                                        SE           1.0                                                                              0.9                                                                              0.8                                                                              0.7                                                                              0.8                                                                              1.0                                                                              0.6                                                                              0.6                                         n.sup.(1)    15 15 15 15 15 15 14 13                                          Progesterone (ng/ml)                                                          Mean         6.7                                                                              6.8                                                                              5.7                                                                              5.8                                                                              5.7                                                                              5.8                                                                              6.3                                                                              6.8                                         SE           0.6                                                                              0.7                                                                              0.6                                                                              0.8                                                                              0.7                                                                              0.4                                                                              0.4                                                                              0.5                                         n.sup.(1)    15 15 15 15 15 15 14 13                                          __________________________________________________________________________     .sup.(1) decreases as a result of pregnancy                              

                                      TABLE 23                                    __________________________________________________________________________    Antibody levels produced by female baboons against Structure XII, baboon      chorionic gonadotropin and                                                    human chorionic gonadotropin after immunization.                              Values were determined from serum samples obtained during the early           luteal phase of each menstrual cycle                                                        Antibody level during menstrual cycle                                         1     2      3      4      5                                    Antibody Reactive To                                                                        moles/liter × 10.sup.-10                                  __________________________________________________________________________    Structure (XII)                                                               Mean          41.4  186.0  205.6  252.0  284.1                                SD            28.6   84.1   67.5  118.7   99.7                                95% CI        (25.6-57.3)                                                                         (139.4-232.5)                                                                        (168.3-243.0)                                                                        (186.1-317.7)                                                                        (226.5-341.7)                        Baboon Chorionic Gonadotropin                                                 Mean           0.7   3.7    5.5    7.7    9.0                                 SD             0.6   3.3    3.4    4.8    4.8                                 95% CI        (0.3-1.0)                                                                           (1.9-5.6)                                                                            (3.6-7.3)                                                                            (5.0-10.4)                                                                           (6.3-11.8)                           Human Chorionic Gonadotropin                                                  Mean          31.9  160.0  172.1  205.5  224.8                                SD            23.9   77.7   55.0  108.2   89.8                                95% CI        (18.7-45.2)                                                                         (117.0-203.1)                                                                        (141.6-202.5)                                                                        (145.5-265.4)                                                                        (172.9-276.6)                        Number of Animals                                                                           15    15     15     15     14                                   __________________________________________________________________________

                  TABLE 24                                                        ______________________________________                                        Comparison of fertility rates for female baboons immunized with               Diphtheria Toxoid with those immunized with conjugate.                        Baboons        Mating Cycle                                                   Immunized With 1      2        3    Totals                                    ______________________________________                                        Diphtheria Toxoid                                                             No. Mated      15     4        1    20                                        No. Pregnant   11     3        0    14                                        Fertility Rate (%)                                                                           73.3   75.0     0    70.0                                      Conjugate                                                                     No. Mated      15     15       14   44                                        No. Pregnant   0      1        1    2                                         Fertility Rate (%)                                                                           0      6.7      7.1  4.6                                       ______________________________________                                    

EXAMPLE XXXVI

This example illustrates the effect of a vaccine of this invention inretarding the growth of Lewis lung carcinoma tumors in mice.

The mice used in these experiments were six-week old mice of the strainC57BL/6J, obtained from the same source as in Example II. The mice weredivided into a test group of 201 animals and a control group of 17animals. The test animals were immunized using the same vaccine asdescribed in the second paragraph of Example V above, each injectioncomprising 200 μg. of conjugate and 100 μg. of adjuvant givensubcutaneously in the lower back. Following the first injection, boosterimmunizations were given 4, 12, and 23 weeks later. The control group ofmice were given immunizations at the same time, but the vaccine usedcontained only vehicle. Two days after the final immunization, eachmouse was inoculated subcutaneously in the lower back with approximatelyone million viable cells of transplatable Lewis lung carcinoma.Measurements of the volume of the tumor were made 10, 14 and 17 daysafter tumor implantation and then, on day 18, the mice were sacrificedand the tumor removed and weighed.

                  TABLE 25                                                        ______________________________________                                        Comparison of tumor volumes and weights in mice implanted with                Lewis Lung Carcinoma following immunization with vehicle or                                                 Mean Tumor                                      Mice    Mean Tumor Volume - cm.sup.3 (±s.e.m.)                                                           Weight in                                       Immunized                                                                             Day of Tumor Growth   gms (±s.e.m.)                                With    10        14        17      Day 18                                    ______________________________________                                        Vehicle 1.89 (0.32)                                                                             5.20 (0.78)                                                                             8.83 (1.14)                                                                           7.00 (0.88)                               Conjugate                                                                             1.23 (0.22)                                                                             2.94 (0.34)*                                                                            5.22 (0.63)*                                                                          4.13 (0.50)*                              ______________________________________                                         *Significantly different (p < 0.05) from vehicle group                   

The results in Table 25 above show that the conjugate-immunized miceshowed significant smaller tumors than the control group, the averagereduction in tumor volume and weight being about 40%.

EXAMPLE XXXVII

This example illustrates the effect of a vaccine of the invention inincreasing the survival rate of mice suffering from viral-inducedleukemia.

The mice used in this experiment were six-week old female mice of SJL/Jstrain. A test group and a control group, each containing approximately20 mice were selected at random and were immunized in the same manner asin Example XXXVI above, the test animals receiving the vaccinecontaining the peptide/diphtheria toxoid conjugate and the control groupreceiving the vaccine containing only the vehicle. However, in thisexperiment a different immunization schedule was followed: the originalimmunization was followed by booster immunizations 4, 9, 20 and 29 weekslater. Six weeks after the first immunization (i.e. two weeks after thesecond immunization) each mouse was injected with approximately 1million Friends' Leukemia Virus and the mice were observed daily fordeaths. The results are shown in Table 26 below and from the results inthis table it will be seen that the vaccine of the invention providedcomplete protection against mortality caused by the leukemia viruswhereas more than 60% of the control group succumbed.

                  TABLE 26                                                        ______________________________________                                        Comparison of survival rates in mice inoculated with Friends'                 Leukemia Virus following immunization with vehicle or conjugate                              Survival Rate - %                                              Days           Group Immunization With                                        After Inoculation                                                                            Vehicle (Control)                                                                         Conjugate                                          ______________________________________                                         0             100.0       100.0                                               22            87.5        100.0                                               29            75.0        100.0                                                98**         75.0        100.0                                              160            56.3        100.0                                               163**         56.3        100.0                                              166            43.8        100.0                                              175            37.5        100.0                                              180            37.5        100.0                                              ______________________________________                                         **Day of booster immunization                                            

EXAMPLE XXXVIII

This example illustrates the effect of a conjugate usable in thevaccines of the invention in retarding the growth of a sarcoma tumor inmice.

Sixty mice of the AKR strain were divided at random into a test groupand a control group each comprising 30 mice. The test group were thenimmunized in the same manner as in Examples XXXVI and XXXVII with avaccine comprising the same conjugate as in those Examples dissolved inFreund's Complete Adjuvant, while the control group simply received theFreund's Adjuvant. Booster immunizations were given to each group threeand seven weeks after the first immunization. Ten days after the thirdimmunization, the mice were inoculated with approximately 2 mm³ ofRidgeway Osteogenic Sarcoma cells, and the mice were observed to see ifthe tumor survived. In the mice in which the tumor survived, tumorvolume measurements were made 22, 26, 29, 31, 33, 38, 40 and 43 daysafter inoculation with the Sarcoma. The average tumor volume in thesurviving mice of each group in which the tumor survived are shown inTable 27 below. The results in Table 27 show that the conjugatesubstantially reduced the rate of tumor growth. The later results shouldbe interpreted with some caution because they are affected by thenumbers of mice surviving; for example, at day 43, seven of theconjugate-immunized mice but only five of the vehicle-immunized micewere surviving and naturally the mice having the largest tumors tendedto succumb first so that the figure for average tumor volume in thevehicle-immunized mice at day 43 is thus artificially reduced by thedeaths of certain mice. Nevertheless, the results in Table 27 do show asignificant reduction in the rate of tumor growth in theconjugate-immunized mice.

The mice were also bled ten days after the final immunization and serumtested for the presence of β-hCG antibodies; all the mice immunized withthe conjugate had produced such antibodies.

                  TABLE 27                                                        ______________________________________                                        Comparison of tumor volumes in mice inoculated with Ridgeway                  Osteogenic Sarcoma following immunization with vehicle or conjugate.                        Mean Tumor Volume - cm.sup.3                                                  Group Immunized With                                            Day of Tumor Growth                                                                           Vehicle (Control)                                                                         Conjugate                                         ______________________________________                                        22              0.15        0.65                                              26              1.15        0.68                                              29              2.30        1.20                                              31              3.45        2.24                                              33              2.33        3.04                                              36              6.27        3.14                                              38              7.55        4.40                                              40              10.36       5.54                                              43              8.21        5.00                                              ______________________________________                                    

EXAMPLE XXXIX

This Example illustrates the preparation and use of a linear polymericpolypeptide of the invention derived from HCG.

Fragment A described above (a fragment having an amino acid sequencecorresponding to the 105-145 sequence of beta-HCG, with a cysteineresidue added to the C-terminal thereof) was polymerized to form ahexamer. A first portion of fragment A had both its thiol groups (on thenon-terminal cysteine) and its non-terminal amino group (on the lysineresidue at the position corresponding to position 122 in beta-HCG)blocked. This blocked form of Fragment A was reacted with thebifunctional organic coupling reagent (or amino group activating agent)MCS in a buffered aqueous solution at pH 6.6, thereby reacting the esterportion of the MCS with the N-terminal amino group of the first portionof Fragment A. The resultant product was then reacted with a secondportion of Fragment A, which was used in the same form as the firstportion of Fragment A except that the C-terminal cysteine bore anunblocked thiol group, thereby reacting the remaining functional groupof the MCS with the free thiol group on the second portion of Fragment Aand producing a dimer in which the N-terminal of the first portion ofFragment A was coupled to the C-terminal of the second portion ofFragment A via an MCS residue. This dimer was then purified by gelfiltration. The polymerization was then repeated in the same manneruntil a hexamer of Fragment A had been produced. Because thepurification following each polymerization step was effected by gelfiltration rather than by reverse-phase, high pressure liquidchromatography, the hexamer was undoubtedly somewhat impure andcontaminated by traces of pentamer, tetramer etc., so that the resultsin the animal tests, results described below, could not be expected tobe as good as would be produced using a pure hexamer.

To test the effectiveness of this hexameric polypeptide in provoking theformation of antibodies to HCG, the hexamer was formed into a vaccineusing Complete Freund's Adjuvant and injected into five rabbits. Eachrabbit was given three injections of the vaccine intramuscularly at 3week intervals, each injection containing 0.5 mg. of the hexamer.Starting three weeks after the first injection, each rabbit was bledweekly and the level of antibodies to HCG in the blood determined. Thefollowing average values of antibody level were found (the figures inparenthesis represent the confidence limits i.e. average+or-standarderror):

                  TABLE 28                                                        ______________________________________                                                         Antibody concentration                                       Weeks After First Injection                                                                    (moles/liters × 10.sup.-10)                            ______________________________________                                        3                5         (2-7)                                              4                18        (13-22)                                            5                30        (21-39)                                            6                45        (30-60)                                            7                58        (32-83)                                            8                61        (34-86)                                            9                77        (50-108)                                           10               110       (50-1/2 125)                                       11               100       (50-1/2 125)                                       12               79        (30-119)                                           13               53        (22-83)                                            ______________________________________                                    

The above results show that even the crude hexamer preparation used inthese experiments was much more strongly immunogenic than the veryweakly immunogenic fragment from which it was derived.

EXAMPLE XL

This Example reports the results of experiments carried out to determinethe immunogenicity and cross-reactivity with LH, of various portions ofthe beta-subunit of HCG.

Synthetic polypeptides corresponding to 12-16 amino acid residueportions of the sequence of beta-HCG were prepared in the same manner asin Example XXXI above. These peptides were then conjugated to diphtheriatoxoid using the coupling techniques described in Example XXXI above. Inall cases, the resultant conjugates contained approximately 30 moleculesof the peptide per 100,000 daltons of diphtheria toxoid. The resultingmodified polypeptide conjugates were then mixed with Complete Freund'sAdjuvant and injected into rabbits using the same techniques as inExample XXXII above. Table 29 shows the results obtained by testing forthe antibody levels to HCG and MLH in these experiments.

                  TABLE 29                                                        ______________________________________                                        Mean Peak Antibody Levels in Sera from Rabbits                                Immunized with Beta-hCG Peptide Conjugates                                    Subunit Sequence                                                                             Antibody levels                                                                          M/L × 15.sup.-10                              ______________________________________                                         1-12          0.92       0.05                                                10-22          0.62       0.05                                                20-32          1.90       0.05                                                30-42          15.70      6.20                                                40-52          1.80       0.05                                                50-62          0.55       0.05                                                60-72          1.78       0.90                                                70-82          4.66       0.05                                                 80-92*                                                                        90-102        1.70       0.85                                                100-112        0.44       0.11                                                110-122        100.45     0.05                                                120-132        3.60       0.05                                                130-145        75.70      0.05                                                ______________________________________                                         *Sequence not tested                                                     

It will be appreciated that numerous changes and modifications can bemade in the embodiments of the invention described above withoutdeparting from the scope of the invention. Accordingly, the foregoingdescription is to be construed in an illustrative and not in alimitative sense, the scope of the invention being defined solely by theappended claims.

I claim:
 1. A method of treating a neoplastic tumor in a mammal whichexpresses thorionic gonadotropin (CG) comprising the stepsof:administering to said mammal an immunologically effective amount of apolypeptide conjugate comprising a polypeptide determinant selected fromthe group consisting of chorionic gonadotropin, a fragment of chorionicgonadotropin and a naturally occurring or synthetic peptide comprisingan amino acid sequence immunologically analogous to at least one epitopeof chorionic gonadotropin, coupled to a carrier biologically foreign tosaid mammal, wherein said polypeptide determinant has the properties of:(a) prior to coupling to said carrier being nonimmunogenic to saidmammal; and (b) after coupling to said carrier, is capable of causingantibodies to form in said mammal which are reactive with chorionicgonadotropin expressed by the cells of said neoplastic tumor.
 2. Themethod of claim 1 wherein said neoplastic tumor is a carcinoma tumor. 3.The method of claim 1 wherein said naturally occurring or syntheticpeptide comprises the amino acid sequence:Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gin-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln.
 4. The method of claim1 wherein said naturally occurring or synthetic peptide comprises theamimo acid sequence: Cys-Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln.
 5. The methodof claim 1, wherein said polypeptide determinant is a synthetic peptidederived from the 105-145 amino acid sequence of the beta subunit ofhuman thorionic gonadotropin and is immunologically analogous to atleast two distinct epitopes of human chorionic gonadotropin.
 6. Themethod of claim 1 wherein said polypeptide determinant is humanchorionic gonadotropin or a fragment thereof.
 7. The method of claim 1wherein said polypeptide conjugate comprises the beta subunit of humanchorionic gonadotropin coupled to tetanus toxoid.
 8. The method of claim1 wherein said polypeptide conjugate comprises a fragment of the betasubunit of human chorionic gonadotropin coupled to tetanus toxoid. 9.The method of claim 1 wherein said carrier is diphtheria toxoid.
 10. Themethod of claim 3 wherein said polypeptide conjugate has the --SH groupof its non-terminal cysteine and the amino group on its lysine blocked.11. A method according to claim 10 wherein said blocking of said --SEgroup is effected with an acetamidomethyl group.
 12. The method of claim4 wherein said carrier is diphtheria toxoid.
 13. The method according toclaim 12 wherein said polypeptide has the --SH group on its non-terminalcysteine blocked.
 14. The method according to claim 13 wherein saidblocking of said --SH group is effected with an acetamidomethyl group.15. The method of claim 1 wherein said polypeptide determinant comprisesan amino acid sequence immunologically analogous to the amino acidresidues 105-145 of the beta subunit of human thorionic gonadotropin.16. The method of claim 11 wherein said polypeptide determinantcomprises an amino acid sequence immunologically analogous to the aminoacid residues 109-145 the beta subunit of human chorionic gonadotropin.17. The method of claim 1 wherein said polypeptide determinant compriseshuman chorionic gonadotropin, a fragment of human chorionicgonadotropin, or a naturally occurring or synthetic peptide comprisingan amino acid sequence immunologically analogous to at least one epitopeof human chorionic gonadotropin and wherein said mammal is a human. 18.The method of claim 17 wherein said carrier is tetanus toxoid.
 19. Themethod of claim 17 wherein said carrier is diphtheria toxoid.
 20. Amethod of treating a neoplastic tumor in a human which expresses humanchorionic gonadotropin comprising the steps of:administering to saidhuman an immunologically effective amount of a polypeptide determinantcomprising a naturally occurring or synthetic peptide including an aminoacid sequence immunologically analogous to at least one epitope of saidhuman chorionic gonadotropin, coupled to a carrier biologically foreignto said human, wherein said polypeptide determinant has the propertiesof: a) prior to coupling to said carrier, being non-immunogenic to saidhuman; and b) after coupling to said carrier, capable of causingantibodies to form in said human which are reactive with human chorionicgonadotropin expressed by the cells of said neoplastic tumor.
 21. Themethod of claim 20, wherein said polypeptide determinant is a syntheticpeptide including an amino acid sequence derived from the 105-145 aminoacid sequence of the beta subunit of human chorionic gonadotropin and isimmunologically analogous to at least two distinct epitopes of humanchorionic gonadotropin.
 22. The method of claim 20 wherein said carrieris diphtheria toxoid.
 23. The method of claim 20 wherein said neoplastictumor is a carcinoma tumor.